Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.

For several decades, the ability of protein synthesis inhibitors (PSI) to suppress the long-term potentiation (LTP) of hippocampal responses is known. It is considered that mechanisms of such impairment are related to a cessation of translation and a delayed depletion of the protein pool required for maintenance of synaptic plasticity. The present study demonstrates that cycloheximide or anisomycin applications reduce amplitudes of the field excitatory postsynaptic potentials as well as the presynaptically mediated form of plasticity, the paired-pulse facilitation after LTP induction in neurons of the CA1 area of hippocampus. We showed that nitric oxide signaling could be one of the pathways that cause the LTP decrease induced by cycloheximide or anisomycin. Inhibitor of the NO synthase, L-NNA or the NO scavenger, PTIO, rescued the late-phase LTP and restored the paired-pulse facilitation up to the control levels. For the first time we have directly measured the nitric oxide production induced by application of the translation blockers in hippocampal neurons using the NO-sensitive dye DAF-FM. Inhibitory analysis demonstrated that changes during protein synthesis blockade downstream the NO signaling cascade are cGMP-independent and apparently are implemented through degradation of target proteins. Prolonged application of the NO donor SNAP impaired the LTP maintenance in the same manner as PSI.

The involvement of protein synthesis in the mechanisms of conditioned food aversion memory impairment and recovery in grape snails was studied. It was found that protein synthesis inhibitor (cycloheximide) injections before a reminder by the conditioned stimulus (CS) caused amnesia development. Three days after amnesia induction, injections of cycloheximide or another protein synthesis inhibitor, anisomycin, combined with a reminder by four CSs resulted in memory retrieval, which was saved for 24 h. Cycloheximide injections and the administration of one CS as a reminder to an amnestic animals caused the memory expression only in response to this CS, while it was absent the next day. The isolated administration of a reminder or inhibitor injections without a reminder was not effective. It is suggested that amnesia is an active process and that one of its mechanisms may be a protein-dependent amnesia reactivation caused by a reminder. The administration of protein synthesis inhibitors led to impairment of amnesia reactivation and to recovery of the state formed before amnesia induction and thus to the recovery of conditioned food aversion memory.

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.

Antimicrobial multidrug resistance and its transmission among strains are serious problems. Success rate is decreased and treatment options are narrowed due to increasing bacterial multidrug resistance. On the other hand, the need for long-term efforts to discover new antibiotics and difficulties finding new treatment protocols make this problem more complex. Combination therapy, especially with synergistic use of antimicrobials is a rational treatment option with huge benefits. Thus, screening antibiotic interactions is crucial for finding better treatment options. Clinicians currently use combinatorial antibiotic treatment as an effective treatment option. However, antibiotics can show synergistic or antagonistic interactions when used together. In our study, we aimed to investigate interactions of antibiotics with different mechanisms of action. Antibiotics, which act as protein synthesis inhibitors (P) and nucleic acid synthesis inhibitors (N) were used in our study. We tested 66 (PN), 15 (NN), and 55 (PP) drug pairs on the Escherichia coli strain. The Loewe additivity model was used and alpha scores were calculated for analysis of interactions of drug combinations. Drug interactions were categorized as synergistic or antagonistic. Accordingly, pairwise combinations of protein synthesis inhibitors (PP) showed stronger synergistic interactions than those of nucleic acid synthesis inhibitors (NN) and nucleic acid synthesis-protein synthesis inhibitors (PN). As a result, the importance of mechanisms of action of drugs is emphasized in the selection of synergistic drug combinations.

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators.

Direct interference with Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 protein-protein interaction (PPI) has recently been introduced as an attractive approach to control life-threatening diseases like myocarditis. The present study aimed to investigate the potential application in myocarditis of a series of novel non-naphthalene derivatives as potential Keap1-Nrf2 PPI inhibitors. Our results indicated that the optimal compound K22 displayed the highest metabolic stability and showed notable Keap1-Nrf2 PPI inhibitory activities in vitro. K22 effectively triggered Nrf2 activation and increased the protein and mRNA expression of Nrf2-regulated genes in H9c2 cells. Moreover, pre-treatment with K22 was shown to mitigate LPS-induced damage to H9c2 cells, causing a marked decrease in the levels of inflammatory factors as well as reactive oxygen species (ROS). Furthermore, K22 was also shown to be non-mutagenic in the Ames test. Overall, our findings suggest that K22 may be a promising drug lead as a Keap1-Nrf2 PPI inhibitor for myocarditis treatment.

Glycogen synthesis was studied in rat hepatocytes isolated by EDTA perfusion. Insulin induced a one and a half to twofold increase in glucose incorporation into glycogen. Insulin stimulated glycogen synthesis was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin (IC50 approximately 40 nM) and LY 294002 (IC50 approximately 20 microM) and the mitogen-activated protein kinase kinase inhibitor PD 98059 (IC50 approximately 40 microM). Wortmannin was without appreciable effect on non-insulin stimulated glycogen synthesis, while LY 294002 and PD 98059 also inhibited the non-insulin stimulated glycogen synthesis. Rapamycin, an inhibitor of p70 ribosomal protein-S6 kinase, was without effect on glycogen synthesis regardless of insulin stimulation.

G protein-coupled receptor kinase 2 (GRK2) is a relevant signaling node of the cellular transduction network, playing major roles in the physiology of various organs/tissues including the heart and blood vessels. Emerging evidence suggests that GRK2 is up regulated in pathological situations such as heart failure, hypertrophy and hypertension, and its inhibition offers a potential therapeutic solution to these diseases. We explored the GRK2 inhibitory activity of a library of cyclic peptides derived from the HJ loop of G protein-coupled receptor kinases 2 (GRK2). The design of these cyclic compounds was based on the conformation of the HJ loop within the X-ray structure of GRK2. One of these compounds, the cyclic peptide 7, inhibited potently and selectively the GRK2 activity, being more active than its linear precursor. In a cellular system, this peptide confirms the beneficial signaling properties of a potent GRK2 inhibitor. Preferred conformations of the most potent analog were investigated by NMR spectroscopy.

Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor 3 (VEGFR3) are tyrosine kinases, which function as key modulators of survival and metastasis signals in cancer cells. Previously, we reported that small molecule chlorpyramine hydrochloride (C4) specifically targets the interaction between FAK and VEGFR3 and exhibits anti-tumor efficacy. In this study, we designed and synthesized a series of 1 (C4) analogs on the basis of structure activity relationship and molecular modeling. The resulting new compounds were evaluated for their binding to the FAT domain of FAK and anti-cancer activity. Amongst all tested analogs, compound 29 augmented anti-proliferative activity in multiple cancer cell lines with stronger binding to the FAT domain of FAK and disrupted the FAK-VEGFR3 interaction. In conclusion, we hope that this work will contribute to further studies of more potent and selective FAK-VEGFR3 protein-protein interaction inhibitors.

Two new families of human farnesyltransferase inhibitors 13a-m and 14a-d, based on a phenothiazine scaffold, were synthesized. Compounds 14a and 14b were the most promising inhibitors of human farnesyltransferase with IC(50) values of 0.7 and 0.6 μM, respectively.

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.

Fatty acid binding protein 4 (FABP4) is a potential drug target for diabetes and atherosclerosis. For discovering new chemical entities as FABP4 inhibitors, structure-based virtual screening (VS) was performed, bioassay demonstrated that 16 of 251 tested compounds are FABP4 inhibitors, among which compound m1 are more active than endogenous ligand linoleic acid (LA). Based on the structure of m1, new derivatives were designed and prepared, leading to the discovery of two more potent inhibitors, compounds 9 and 10. To further explore the binding mechanisms of these new inhibitors, we determined the X-ray structures of the complexes of FABP4-9 and FABP4-10, which revealed similar binding conformations of the two compounds. Residue Ser53 and Arg126 formed direct hydrogen bonding with the ligands. We also found that 10 could significantly reduce the levels of lipolysis on mouse 3T3-L1 adipocytes. Taken together, in silico, in vitro and crystallographic data provide useful hints for future development of novel inhibitors against FABP4.

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

Protein synthesis is regulated by the availability of amino acids, the engagement of growth factor signaling pathways, and adenosine triphosphate (ATP) levels sufficient to support translation. Crosstalk between these inputs is extensive, yet other regulatory mechanisms remain to be characterized. For example, the translation initiation inhibitor rocaglamide A (RocA) induces thioredoxin-interacting protein (TXNIP). TXNIP is a negative regulator of glucose uptake; thus, its induction by RocA links translation to the availability of glucose. MondoA is the principal regulator of glucose-induced transcription, and its activity is triggered by the glycolytic intermediate, glucose 6-phosphate (G6P). MondoA responds to G6P generated by cytoplasmic glucose and mitochondrial ATP (mtATP), suggesting a critical role in the cellular response to these energy sources. TXNIP expression is entirely dependent on MondoA; therefore, we investigated how protein synthesis inhibitors impact its transcriptional activity.

Protein arginine methyltransferase 1 (PRMT1) is involved in many biological activities, such as gene transcription, signal transduction, and RNA processing. Overexpression of PRMT1 is related to cardiovascular diseases, kidney diseases, and cancers; therefore, selective PRMT1 inhibitors serve as chemical probes to investigate the biological function of PRMT1 and drug candidates for disease treatment. Our previous work found trimethine cyanine compounds that effectively inhibit PRMT1 activity. In our present study, we systematically investigated the structure-activity relationship of cyanine structures. A pentamethine compound, E-84 (compound 50), showed inhibition on PRMT1 at the micromolar level and 6- to 25-fold selectivity over CARM1, PRMT5, and PRMT8. The cellular activity suggests that compound 50 permeated the cellular membrane, inhibited cellular PRMT1 activity, and blocked leukemia cell proliferation. Additionally, our molecular docking study suggested compound 50 might act by occupying the cofactor binding site, which provided a roadmap to guide further optimization of this lead compound.

Insulin stimulates glucose uptake in adipocytes by triggering translocation of glucose transporter 4-containg vesicles to the plasma membrane. Under basal conditions, these vesicles (IRVs for insulin-responsive vesicles) are retained inside the cell via a "static" or "dynamic" mechanism. We have found that inhibitors of RNA and protein synthesis, actinomycin D and emetine, stimulate Glut4 translocation and glucose uptake in adipocytes without engaging conventional signaling proteins, such as Akt, TBC1D4, or TUG. Actinomycin D does not significantly affect endocytosis of Glut4 or recycling of transferrin, suggesting that it specifically increases exocytosis of the IRVs. Thus, the intracellular retention of the IRVs in adipocytes requires continuous RNA and protein biosynthesis de novo. These results point out to the existence of a short-lived inhibitor of IRV translocation thus supporting the "static" model.

The β-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential target for the suppression of hyperactive Wnt/β-catenin signaling that is vigorously involved in cancer initiation and development. Herein, we first described quercetin and its derivatives had potential inhibitory effects on β-catenin/BCL9 PPI. The most potent compound, quercetin-3'-O-(4-methylpiperazine-1-yl) propyl (C1), directly binded with β-catenin and disrupted the β-catenin/BCL9 interaction in both the protein level and the cellular context. C1 also effectively inhibited colorectal cancer in vitro and showed better selectivity in inhibiting hyperactive Wnt/β-catenin signaling cells like CT26 and HCT116. And we further confirmed that C1 could inhibit CT26 tumor growth in vivo and regulate the tumor immune microenvironment. This study provides a good chemical probe to explore β-catenin-related biology and a drug-like quercetin derivative as novel β-catenin/BCL9 PPI inhibitors for further drug development.

CK2 and PIM-1 are serine/threonine kinases involved in the regulation of many essential processes, such as proliferation, differentiation, and apoptosis. Inhibition of CK2 and PIM-1 kinase activity has been shown to significantly reduce the viability of cancer cells by inducing apoptosis. A series of novel amino alcohol derivatives of parental DMAT were designed and synthesized as potent dual CK2/PIM-1 inhibitors. Concomitantly with the inhibition studies toward recombinant CK2 and PIM-1, the influence of the obtained compounds on the viability of three human carcinoma cell lines, i.e., acute lymphoblastic leukemia (CCRF-CEM), human chronic myelogenous leukemia (K-562), and breast cancer (MCF-7), as well as non-cancerous cells (Vero), was evaluated using an MTT assay. Induction of apoptosis and cell cycle progression after treatment with the most active compound and a lead compound were studied by flow-cytometry-based assay. Additionally, autophagy induction in K-562 cells and intracellular inhibition of CK2 and PIM-1 in all the tested cell lines were evaluated by qualitative/quantitative fluorescence-based assay and Western blot method, respectively. Among the newly developed inhibitors, 1,1,1-trifluoro-3-[(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)amino]propan-2-ol demonstrates the highest selectivity and the most prominent proapoptotic properties towards the studied cancer cells, especially towards acute lymphoblastic leukemia, in addition to inducing autophagy in K-562 cells.