Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus.

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.

Optimization of the antimicrobial peptide Bac7 by deep mutational scanning.

BACKGROUND: Intracellularly active antimicrobial peptides are promising candidates for the development of antibiotics for human applications. However, drug development using peptides is challenging as, owing to their large size, an enormous sequence space is spanned. We built a high-throughput platform that incorporates rapid investigation of the sequence-activity relationship of peptides and enables rational optimization of their antimicrobial activity. The platform is based on deep mutational scanning of DNA-encoded peptides and employs highly parallelized bacterial self-screening coupled to next-generation sequencing as a readout for their antimicrobial activity. As a target, we used Bac71-23, a 23 amino acid residues long variant of bactenecin-7, a potent translational inhibitor and one of the best researched proline-rich antimicrobial peptides. RESULTS: Using the platform, we simultaneously determined the antimicrobial activity of >600,000 Bac71-23 variants and explored their sequence-activity relationship. This dataset guided the design of a focused library of ~160,000 variants and the identification of a lead candidate Bac7PS. Bac7PS showed high activity against multidrug-resistant clinical isolates of E. coli, and its activity was less dependent on SbmA, a transporter commonly used by proline-rich antimicrobial peptides to reach the cytosol and then inhibit translation. Furthermore, Bac7PS displayed strong ribosomal inhibition and low toxicity against eukaryotic cells and demonstrated good efficacy in a murine septicemia model induced by E. coli. CONCLUSION: We demonstrated that the presented platform can be used to establish the sequence-activity relationship of antimicrobial peptides, and showed its usefulness for hit-to-lead identification and optimization of antimicrobial drug candidates.

The Yeast Permease Agp2 Senses Cycloheximide and Undergoes Degradation That Requires the Small Protein Brp1-Cellular Fate of Agp2 in Response to Cycloheximide.

The Saccharomyces cerevisiae Agp2 is a plasma membrane protein initially reported to be an uptake transporter for L-carnitine. Agp2 was later rediscovered, together with three additional proteins, Sky1, Ptk2, and Brp1, to be involved in the uptake of the polyamine analogue bleomycin-A5, an anticancer drug. Mutants lacking either Agp2, Sky1, Ptk2, or Brp1 are extremely resistant to polyamines and bleomycin-A5, suggesting that these four proteins act in the same transport pathway. We previously demonstrated that pretreating cells with the protein synthesis inhibitor cycloheximide (CHX) blocked the uptake of fluorescently labelled bleomycin (F-BLM), raising the possibility that CHX could either compete for F-BLM uptake or alter the transport function of Agp2. Herein, we showed that the agp2Δ mutant displayed striking resistance to CHX as compared to the parent, suggesting that Agp2 is required to mediate the physiological effect of CHX. We examined the fate of Agp2 as a GFP tag protein in response to CHX and observed that the drug triggered the disappearance of Agp2 in a concentration- and time-dependent manner. Immunoprecipitation analysis revealed that Agp2-GFP exists in higher molecular weight forms that were ubiquitinylated, which rapidly disappeared within 10 min of treatment with CHX. CHX did not trigger any significant loss of Agp2-GFP in the absence of the Brp1 protein; however, the role of Brp1 in this process remains elusive. We propose that Agp2 is degraded upon sensing CHX to downregulate further uptake of the drug and discuss the potential function of Brp1 in the degradation process.

Learning against the Background of DNA Methyltransferase Inhibition Leads to the Formation of Memory That Is Resistant to Reactivation and Impairment.

The involvement of DNA methylation in the mechanisms of formation of conditioned food aversion memory was studied on Helix lucorum snails. The dynamics of aversion formation in snails injected with DNA methyltransferase inhibitor RG108 did not differ from that in control snails. The memory was retained for more than one month after training following RG108 injection and the duration of memory persistence did not differ from that in control animals. However, the characteristics of memory in control and experimental snails differed significantly. In control snails, injections of glutamate NMDA-receptor antagonist or protein synthesis inhibitor before memory retrieval caused disorders in the memory reconsolidation and development of amnesia 2 days after training. By contrast, injections of these substances before retrieval to snails trained against the background of RG108 treatment caused no memory disorders. We hypothesized that inhibition of DNA methylation processes led to the formation of strong memory, not reactivated after retrieval and not transformed into a labile state sensitive to amnesic agents.

Identification of nagilactone E as a protein synthesis inhibitor with anticancer activity.

Norditerpenoids and dinorditerpenoids represent diterpenoids widely distributed in the genus Podocarpus with notable chemical structures and biological activities. We previously reported that nagilactone E (NLE), a dinorditerpenoid isolated from Podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. In this study we investigated the in vivo effect of NLE against lung cancer as well as the underlying mechanisms. We administered NLE (10 mg·kg-1·d-1, ip) to CB-17/SCID mice bearing human lung cancer cell line A549 xenograft for 3 weeks. We found that NLE administration significantly suppressed the tumor growth without obvious adverse effects. Thereafter, RNA sequencing (RNA-seq) analysis was performed to study the mechanisms of NLE. The effects of NLE on A549 cells have been illustrated by GO and pathway enrichment analyses. CMap dataset analysis supported NLE to be a potential protein synthesis inhibitor. The inhibitory effect of NLE on synthesis of total de novo protein was confirmed in Click-iT assay. Using the pcDNA3-RLUC-POLIRES-FLUC luciferase assay we further demonstrated that NLE inhibited both cap-dependent and cap-independent translation. Finally, molecular docking revealed the low-energy binding conformations of NLE and its potential target RIOK2. In conclusion, NLE is a protein synthesis inhibitor with anticancer activity.

Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps.

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics.

Peculiarities in Synthesis of Proteins Implicated in Memory Reconsolidation and Induction of Amnesia.

The peculiarities of implication of NMDA glutamate receptors and protein synthesis into the mechanisms responsible for impairment of memory reconsolidation were studied in edible snails conditioned to food aversion. Injections of NMDA glutamate receptor antagonists or protein synthesis inhibitor prior to reminding with conditioned food stimulus provoked development of amnesia after different latent periods. NMDA glutamate receptor antagonists gradually weakened the aversive reactions to conditioned stimulus presented in parallel with reminder during 1 h. The protein synthesis inhibitor cycloheximide provoked the development of amnesia only 2.5 h after the onset of reminding procedure. Combined injections of protein synthesis inhibitor and NMDA glutamate receptor antagonist prior to reminding completely prevented the development of amnesia. The data agree with hypothesis that memory reconsolidation and amnesia are distinct processes, which need activation of synthesis of specific proteins.

Fragments of the Nonlytic Proline-Rich Antimicrobial Peptide Bac5 Kill Escherichia coli Cells by Inhibiting Protein Synthesis.

Unlike most antimicrobial peptides (AMPs), the main mode of action of the subclass of proline-rich antimicrobial peptides (PrAMPs) is not based on disruption of the bacterial membrane. Instead, PrAMPs exploit the inner membrane transporters SbmA and YjiL/MdtM to pass through the bacterial membrane and enter the cytosol of specific Gram-negative bacteria, where they exert an inhibitory effect on protein synthesis. Despite sharing a high proline and arginine content with other characterized PrAMPs, the PrAMP Bac5 has a low sequence identity with them. Here we investigated the mode of action of three N-terminal Bac5 fragments, Bac5(1-15), Bac5(1-25), and Bac5(1-31). We show that Bac5(1-25) and Bac5(1-31) retained excellent antimicrobial activity toward Escherichia coli and low toxicity toward eukaryotic cells, whereas Bac5(1-15) was inactive. Bac5(1-25) and Bac5(1-31) inhibited bacterial protein synthesis in vitro and in vivo Competition assays suggested that the binding site of Bac5 is within the ribosomal tunnel, where it prevents the transition from the initiation to the elongation phase of translation, as reported for other PrAMPs, such as the bovine PrAMP Bac7. Surprisingly, unlike Bac7, Bac5(1-25) exhibited species-specific inhibition, being an excellent inhibitor of protein synthesis on E. coli ribosomes but a poor inhibitor on Thermus thermophilus ribosomes. This indicates that while Bac5 most likely has an overlapping binding site with Bac7, the mode of interaction is distinct, suggesting that Bac5 fragments may be interesting alternative lead compounds for the development of new antimicrobial agents.

Anterograde Amnesia Induced by Disruption of Consolidation or Reconsolidation of Long-Term Memory.

Mechanisms of amnesia caused by impairment of consolidation or reconsolidation of conditioned food aversion memory with protein synthesis inhibitor cycloheximide were studied in Helix lucorum. Cycloheximide injection during training or memory reconsolidation in trained snails produced amnesia. In both cases, repeated training 10 days after amnesia induction led to short-term memory formation, while long-term memory was not formed, despite the fact that the number of conditioned and reinforcing stimuli combinations was higher than during initial training. The possibility of formation of short-term memory not transforming into long-term memory is one of the key characteristics of anterograde amnesia. Our findings data and experimental model can be used for analysis of specific molecular mechanisms of anterograde amnesia.

Intrahippocampal Anisomycin Impairs Spatial Performance on the Morris Water Maze.

New memories are thought to be solidified (consolidated) by de novo synthesis of proteins in the period subsequent to learning. This view stems from the observation that protein synthesis inhibitors, such as anisomycin (ANI), administered during this consolidation period cause memory impairments. However, in addition to blocking protein synthesis, intrahippocampal infusions of ANI cause the suppression of evoked and spontaneous neural activity, suggesting that ANI could impair memory expression by simply preventing activity-dependent brain functions. Here, we evaluated the influence of intrahippocampal ANI infusions on allocentric spatial navigation using the Morris water maze, a task well known to require dorsal hippocampal integrity. Young, adult male Sprague Dawley rats were implanted with bilateral dorsal hippocampal cannulae, and their ability to learn the location of a hidden platform was assessed before and following infusions of ANI, TTX, or vehicle (PBS). Before infusion, all groups demonstrated normal spatial navigation (training on days 1 and 2), whereas 30 min following infusions (day 3) both the ANI and TTX groups showed significant impairments in allocentric navigation, but not visually cued navigation, when compared with PBS-treated animals. Spatial navigational deficits appeared to resolve on day 4 in the ANI and TTX groups, 24 h following infusion. These results show that ANI and TTX inhibit the on-line function of the dorsal hippocampus in a similar fashion and highlight the importance of neural activity as an intervening factor between molecular and behavioral processes. SIGNIFICANCE STATEMENT: The permanence of memories has long thought to be mediated by the production of new proteins, because protein synthesis inhibitors can block retrieval of recently learned information. However, protein synthesis inhibitors may have additional detrimental effects on neurobiological function. Here we show that anisomycin, a commonly used protein synthesis inhibitor in memory research, impairs on-line brain function in a way similar to an agent that eliminates electrical neural activity. Since disruption of neural activity can also lead to memory loss, it may be that memory permanence is mediated by neural rehearsal following learning.

Final analysis of the efficacy and safety of omacetaxine mepesuccinate in patients with chronic- or accelerated-phase chronic myeloid leukemia: Results with 24 months of follow-up.

BACKGROUND: Omacetaxine, a protein synthesis inhibitor, is indicated in the United States for the treatment of patients with chronic-phase (CP) or accelerated-phase (AP) chronic myeloid leukemia (CML) with resistance and/or intolerance to 2 or more tyrosine kinase inhibitors. METHODS: The final analysis, with 24 months of follow-up, included additional efficacy and safety analyses to assess the benefit of long-term omacetaxine administration (1.25 mg/m(2) twice daily for 14 days every 28 days followed by 7 days every 28 days) in CP-CML and AP-CML patients receiving >3 cycles. RESULTS: Eighteen percent of CP-CML patients achieved a major cytogenetic response (MCyR) with a median duration of 12.5 months (95% confidence interval [CI], 3.5 months to not reached [NR]); responses were maintained for ≥12 months in 3 of 14 responders, and the median overall survival (OS) was 40.3 months (95% CI, 23.8 months to NR). Among patients with AP-CML, 14% achieved or maintained a major hematologic response for a median of 4.7 months (95% CI, 3.6 months to NR); MCyR was not achieved, and the median OS was 14.3 months (95% CI, 6.7-18.7 months). In patients with CP-CML and patients with AP-CML who received >3 cycles of treatment (n = 50 and n = 14, respectively), the median OS was 49.3 months (95% CI, 23.8 months to NR) and 24.6 months (95% CI, 12-37.2 months), respectively. Grade 3 or higher hematologic toxicities were the major side effects (79% and 73% for CP-CML and AP-CML, respectively), with discontinuation due to toxicity in 10% of CP patients and in 5% of AP patients. CONCLUSIONS: These results suggest that the long-term administration of omacetaxine is feasible with dose adjustments to manage toxicities and that omacetaxine provides a durable benefit for some patients.

Effects of linezolid on suppressing in vivo production of staphylococcal toxins and improving survival outcomes in a rabbit model of methicillin-resistant Staphylococcus aureus necrotizing pneumonia.

BACKGROUND: Linezolid is recommended for treatment of pneumonia and other invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The premise underlying this recommendation is that linezolid inhibits in vivo production of potent staphylococcal exotoxins, including Panton-Valentine leukocidin (PVL) and α-hemolysin (Hla), although supporting evidence is lacking. METHODS: A rabbit model of necrotizing pneumonia using MRSA clone USA300 was used to compare therapeutic effects of linezolid (50 mg/kg 3 times/day) and vancomycin (30 mg/kg 2 times/day) administered 1.5, 4, and 9 hours after infection on host survival outcomes and in vivo bacterial toxin production. RESULTS: Mortality rates were 100% for untreated rabbits and 83%-100% for vancomycin-treated rabbits. In contrast, mortality rates were 25%, 50%, and 100% for rabbits treated with linezolid 1.5, 4, and 9 hours after infection, respectively. Compared with untreated and vancomycin-treated rabbits, improved survival of rabbits treated 1.5 hours after infection with linezolid was associated with a significant decrease in bacterial counts, suppressed bacterial production of PVL and Hla, and reduced production of the neutrophil-chemoattractant interleukin 8 in the lungs. CONCLUSIONS: Across the study interval, only early treatment with linezolid resulted in significant suppression of exotoxin synthesis and improved survival outcomes in a rabbit model of MRSA necrotizing pneumonia.

Preclinical evaluation of protein synthesis inhibitor omacetaxine in pediatric brainstem gliomas.

Background: Diffuse intrinsic pontine gliomas (DIPGs) pose a significant challenge as a highly aggressive and currently incurable form of pediatric brain cancer, necessitating the development of novel therapeutic strategies. Omacetaxine, an FDA-approved protein synthesis inhibitor for treating certain hematological malignancies, was investigated for its potential antitumor effects against preclinical DIPG models. Methods: We employed primary DIPG cultures to study omacetaxine's cytotoxicity and its impact on colony formation. Annexin V staining and flow cytometry assessed apoptosis. Wound healing assays evaluated migration, while western blotting determined inhibition of oncogenic proteins. We tested omacetaxine's therapeutic efficacy in an orthotopic DIPG model and assessed brain penetration using mass spectrometry. Results: We found a pronounced cytotoxic activity of omacetaxine against DIPG neurospheres, with low IC50 values of approximately 20 nM. Omacetaxine exerted its anti-proliferative effect by inhibiting protein synthesis and the induction of apoptotic pathways, evidenced by significant elevated levels of cleaved caspase 3 and cleaved PARP, both key markers of apoptosis. Omacetaxine effectively targeted oncogenic players such as PDGFRα and PI3K without additional effects on the mTOR signaling pathway. Furthermore, our study revealed the inhibitory effects of omacetaxine on cell migration, and a significant reduction in integrin/FAK signaling, which plays a crucial role in tumor progression and metastasis. Conclusions: Despite these promising in vitro effects, omacetaxine's efficacy in an orthotopic DIPG model was limited due to inadequate penetration across the blood-brain barrier. As such, further research and advancements are crucial to improve the drug's brain penetration, thus enhancing its overall therapeutic potential.

Licochalcone A inhibits interferon-gamma-induced programmed death-ligand 1 in lung cancer cells.

BACKGROUND: Programmed death-ligand 1 (PD-L1), which can be induced by interferon-gamma (IFN-γ) in the tumor microenvironment, is a critical immune checkpoint in cancer immunotherapy. Natural products which reduce IFN-γ-induced PD-L1 might be exert immunotherapy effect. Licochalcone A (LCA), a natural compound derived from the root of Glycyrrhiza inflata Batalin. (Fabaceae), was found to interfere IFN-γ-induced PD-L1. PURPOSE: The aim of this study is to further clarify the effect and the mechanism of LCA on inhibiting IFN-γ-induced PD-L1 in lung cancer cells. METHODS: The expression levels of PD-L1 were evaluated by flow cytometry, western blot and qRT-PCR. Click-iT protein synthesis assay and luciferase assay were used to identify the effect of LCA on protein synthesis. Jurkat T cell proliferation and apoptosis in the co-culture system were detected by flow cytometry. Flow cytometry was also applied to evaluate reactive oxygen species (ROS) generation. RESULTS: LCA downregulated IFN-γ-induced PD-L1 protein expression and membrane localization in human lung cancer cells, regardless of inhibiting PD-L1 mRNA level or promoting its protein degradation. LCA decreased apoptosis and proliferative inhibition of Jurkat T cells caused by IFN-γ-induced PD-L1-expressing in A549 cells in the co-culture system. Strikingly, LCA was verified as a protein synthesis inhibitor, which reduced both cap-dependent and -independent translation. LCA inhibited PD-L1 translation, likely due to inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α pathway. Furthermore, LCA induced ROS generation in a time-dependent manner in lung cancer cells. N-acetyl-L-cysteine (NAC) not only revered ROS generation triggered by LCA but also restored IFN-γ-induced expression of PD-L1. Both the inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α axis triggered by LCA was restored by co-treatment with NAC. CONCLUSION: LCA abrogated IFN-γ-induced PD-L1 expression via ROS generation to abolish the protein translation, indicating that LCA has the potential to be applied in cancer immunotherapy.

Structure and Function of the Bacterial Protein Toxin Phenomycin.

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime toward mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell-free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin, however, have remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure that is critical for the toxicity of phenomycin.

Protein synthesis inhibition promotes nitric oxide generation and activation of CGKII-dependent downstream signaling pathways in the retina.

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine "pools" in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.

Ants learn fast and do not forget: associative olfactory learning, memory and extinction in Formica fusca.

Learning is a widespread phenomenon that allows behavioural flexibility when individuals face new situations. However, learned information may lose its value over time. If such a memory endures, it can be deleterious to individuals. The process of extinction allows memory updating when the initial information is not relevant anymore. Extinction is widespread among animals, including humans. We investigated associative appetitive learning in an ant species that is widely distributed in the Northern Hemisphere, Formica fusca. We studied acquisition and memory between 1 h and one week after conditioning, as well as the extinction process. Ants learn very rapidly, their memory lasts up to 3 days, decreases slowly over time and is highly resistant to extinction, even after a single conditioning trial. Using a pharmacological approach, we show that this single-trial memory critically depends on protein synthesis (long-term memory). These results indicate that individual ant workers of F. fusca show remarkable learning and memory performances. Intriguingly, they also show a strong resistance to updating learned associations. Resistance to extinction may be advantageous when the environment is stochastic and individuals need to switch often from one learned task to another.

Omadacycline: A Newly Approved Antibacterial from the Class of Tetracyclines.

Omadacycline (Nuzyra®) is a new aminomethylcycline, approved by the U. S. Food and Drug Administration in 2018, as a tetracycline antibacterial. It can be used in community-acquired pneumonia and in acute bacterial skin and skin-structure infections. It was developed and is commercialized by Paratek Pharmaceuticals. It is a semisynthetic compound, derived from minocycline, capable of evading widely distributed efflux and target protection antibacterial resistance mechanisms and has demonstrated activity in a broad spectrum of bacteria.

High-Throughput Screening for Protein Synthesis Inhibitors Targeting Aminoacyl-tRNA Synthetases.

Aminoacylation has been implicated in a wide variety of cancers. Aminoacyl-tRNA synthetases (ARSs) exist in large excess in tumor cells due to their increased demand for translation, whereas most other protein-synthesis apparatuses are quantitatively limited. Among other components that constitute the translation machinery-namely, tRNA, amino acid, ATP, and ARS-ARS is the only target that can be blocked by small molecules. No constitutively active ARSs have been reported, and mutations of ARS can cause inaccurate substrate recognition and malformation of the multi-ARS complex (MSC). Hence, interference of the activity is expected to be independent of genotype without developing resistance. Here, we report a high-throughput screening (HTS) system to find mammalian ARS inhibitors. The rabbit-reticulocyte lysate we used closely resembles both the individual and complexed structures of human ARSs, and it may predispose active compounds that are readily applicable for humankind. This assay was further validated because it identified familiar translational inhibitors from a pilot screen, such as emetine, proving its suitability for our purpose. The assay demonstrated excellent quality control (QC) parameters and reproducibility, and is proven ready for further HTS campaigns with large chemical libraries.

Incompatibility of chemical protein synthesis inhibitors with accurate measurement of extended protein degradation rates.

Protein synthesis inhibitors are commonly used for measuring protein degradation rates, but may cause cytotoxicity via direct or indirect mechanisms. This study aimed to identify concentrations providing optimal inhibition in the absence of overt cytotoxicity. Actinomycin D, cycloheximide, emetine, and puromycin were assessed individually, and in two-, three-, and four-drug combinations for protein synthesis inhibition (IC50 ) and cytotoxicity (CC50 ) over 72 h. Experiments were conducted in HepG2 cells and primary rat hepatocytes (PRH). IC50 for actinomycin D, cycloheximide, emetine, and puromycin were 39 ± 7.4, 6600 ± 2500, 2200 ± 1400, and 1600 ± 1200 nmol/L; with corresponding CC50 values of 6.2 ± 7.3, 570 ± 510, 81 ± 9, and 1300 ± 64 nmol/L, respectively, in HepG2 cells. The IC50 were 1.7 ± 1.8, 290 ± 90, 620 ± 920, and 2000 ± 2000 nmol/L, with corresponding CC50 values of 0.98 ± 1.8, 680 ± 1300, 180 ± 700, and 1600 ± 1000 (SD) nmol/L, respectively, in PRH. CC50 were also lower than the IC50 for all drug combinations in HepG2 cells. These data indicate that using pharmacological interference is inappropriate for measuring protein degradation over a protracted period, because inhibitory effects cannot be extricated from cytotoxicity.