Assessment of translation rate in leukemic cells and immune cells of the microenvironment by OPP protein synthesis assay.

Despite being tightly regulated, messenger RNA (mRNA) translation, a manner in which cells control expression of genes and rapidly respond to stimuli, is highly dysfunctional and plastic in pathologies including cancer. Conversely, the investigation of molecular mechanisms whereby mRNA translation becomes aberrant in cancer, as well as inhibition thereof, become critical in developing novel therapeutic approaches. More specifically, in malignancies such as chronic lymphocytic leukemia in which aberrant global and transcript specific translation has been linked with poorer patient outcomes, targeting translation is a relevant approach, with various translation inhibitors under development. Here we elaborate on a protein synthesis assay by flow cytometry, O-propargyl-puromycin, demonstrating global mRNA translation rate with a variety of different applications including cell lines, primary cells or co-culture systems in vitro. This method provides a comprehensive tool in quantifying the rate of global mRNA translation in cancer cells, as well as that of the tumor microenvironment cells, or in response to inhibitory therapeutic agents while offering the possibility to simultaneously assess other cellular markers.

Chemical inhibition of the integrated stress response impairs the ubiquitin-proteasome system.

Inhibitors of the integrated stress response (ISR) have been used to explore the potential beneficial effects of reducing the activation of this pathway in diseases. As the ISR is in essence a protective response, there is, however, a risk that inhibition may compromise the cell's ability to restore protein homeostasis. Here, we show that the experimental compound ISRIB impairs degradation of proteins by the ubiquitin-proteasome system (UPS) during proteotoxic stress in the cytosolic, but not nuclear, compartment. Accumulation of a UPS reporter substrate that is intercepted by ribosome quality control was comparable to the level observed after blocking the UPS with a proteasome inhibitor. Consistent with impairment of the cytosolic UPS, ISRIB treatment caused an accumulation of polyubiquitylated and detergent insoluble defective ribosome products (DRiPs) in the presence of puromycin. Our data suggest that the persistent protein translation during proteotoxic stress in the absence of a functional ISR increases the pool of DRiPs, thereby hindering the efficient clearance of cytosolic substrates by the UPS.

An Enhanced Retroviral Vector for Efficient Genetic Manipulation and Selection in Mammalian Cells.

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies.

A high content imaging assay for identification of specific inhibitors of native Plasmodium liver stage protein synthesis.

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be capable of killing parasites in their liver and blood stage forms, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis, as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits; both of which are parasite-specific quinoline-4-carboxamides, and analogs of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for the specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing the identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity.

Integrated transcriptomics- and structure-based drug repositioning identifies drugs with proteasome inhibitor properties.

Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the ß5 subunit, our in vitro study revealed that these compounds inhibited the ß1, ß2, and ß5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors.

High-throughput drug screening using a library of antibiotics targeting cancer cell lines that are resistant and sensitive to gemcitabine.

Gemcitabine is a nucleoside analog widely used as an anticancer agent against several types of cancer. Although gemcitabine sometimes shows excellent effectiveness, cancer cells are often poorly responsive to or resistant to the drug. Recently, specific strains or dysbiosis of the human microbiome were correlated with drug reactivity and resistance acquisition. Therefore, we aimed to identify antibiotic compounds that can modulate the microbiome to enhance the responsiveness to gemcitabine. To achieve this, we confirmed the gemcitabine responsiveness based on public data and conducted drug screening on a set of 250 antibiotics compounds. Subsequently, we performed experiments to investigate whether the selected compounds could enhance the responsiveness to gemcitabine. First, we grouped a total of seven tumor cell lines into resistant and sensitive group based on the IC50 value (1 µM) of gemcitabine obtained from the public data. Second, we performed high-throughput screening with compound treatments, identifying seven compounds from the resistant group and five from the sensitive group based on dose dependency. Finally, the combination of the selected compound, puromycin dihydrochloride, with gemcitabine in gemcitabine-resistant cell lines resulted in extensive cell death and a significant increase in cytotoxic efficacy. Additionally, mRNA levels associated with cell viability and stemness were reduced. Through this study, we screened antibiotics to further improve the efficacy of existing anticancer drugs and overcome resistance. By combining existing anticancer agents and antibiotic substances, we hope to establish various drug combination therapies and ultimately improve cancer treatment efficacy.

Angiotensin II-independent abnormal renal vascular reactivity during puromycin nephropathy.

Experimental glomerulonephritis results in hypertension that is sensitive to salt. Nevertheless, salt retention alone cannot explain the increase in blood pressure. Angiotensin antagonistic therapy reduces hypertension caused by puromycin amino nucleosides (PAN). We investigated the hypothesis that PAN modifies renal vascular reactivity through processes dependent on angiotensin. Long-Evans rats were given an intraperitoneal injection of either puromycin (150 mg/kg) or saline (controls). Group 1 was fed a normal sodium diet (NSD, n = 9). Group 2 was given 30 mg/L of quinapril (Q) in addition to NSD (NSD + Q; n = 6). Group 3 received a high sodium diet (HSD, n = 7), and Group 4 received HSD + Q (n = 7). Systolic blood pressure (SBP), plasma creatinine, proteinuria, and sodium balance were monitored for 12 days. On day 15, renal vascular reactivity was assessed by administering increasing doses of angiotensin II, acetylcholine (ACh), and sodium nitroprusside (SNP) directly into the renal artery. SBP progressively increased in all PAN groups. This increase in SBP was greater in the HSD groups and was not significantly altered by Q treatment. SBP increased by 22 ± 4% (NSD), 51 ± 5% (NSD + Q), 81 ± 10% (HSD), and 65 ± 8% (HSD + Q). The renal blood flow of PAN rats did not return to baseline despite their normal renal vasoconstrictor responses to angiotensin II. Additionally, they showed reduced renal vasodilator responses to SNP and Ach. The vasodilator responses to both vasodilators were surprisingly unaffected by the inhibition of the angiotensin-converting enzyme (ACE). Renal vasodilator responses to both endothelium-dependent and independent variables were reduced in early PAN-induced hypertension. We found that the angiotensin-mediated mechanism is not responsible for this altered renal vasoreactivity.

Modulation of endogenous opioid signaling by inhibitors of puromycin-sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Enrichment of prime-edited mammalian cells with surrogate PuroR reporters.

Prime editing is a programmable genetic method that can precisely generate any desired small-scale variations in cells without requiring double-strand breaks and DNA donors. However, higher editing efficiency is greatly desirable for wide practical applications. In this study, we developed a target-specific prime editing reporter (tsPER) and a universal prime editing reporter (UPER) to facilitate rapid selection of desired edited cells through puromycin screening. The modification efficiency of HEK3_i1CTT_d5G in HEK293T cells improved from 36.37 % to 64.84 % with the incorporation of tsPER. The target sequence of interested genes could be custom inserted into a selection cassette in tsPER to establish personalized reporters. The UPER demonstrated PE3 editing efficiency up to 74.49 % on HEK3_i1CTT_d5G and 73.52 % on HEK3_i1His6, achieved through co-selection with an additional pegRNA (puro) to repair the mutant PuroR cassette. Overall, tsPER and UPER robustly improved the efficiency of prime editing. Both of these approaches expand enrichment strategies for genomically modified cells and accelerate the generation of genetically modified models.

Development of a universal antibiotic resistance screening system for efficient enrichment of C-to-G and A-to-G base editing.

To expand the scope of genomic editing, a C-to-G transversion-based editor called CGBE has been developed for precise single-nucleotide genomic editing. However, limited editing efficiency and product purity have hindered the development and application of CGBE. In this study, we introduced the Puromycin-Resistance Screening System, referred to as CGBE/ABE-PRSS, to select genetically modified cells via the CGBE or ABE editors. The CGBE/ABE-PRSS system significantly improves the enrichment efficiency of CGBE- or ABE-modified cells, showing enhancements of up to 59.6 % compared with the controls. Our findings indicate that the CGBE/ABE-PRSS, when driven by the CMV promoter, results in a higher enrichment of edited cells compared to the CAG and EF1α promoters. Furthermore, we demonstrate that this system is compatible with different versions of both CGBE and ABE, enabling various cell species and simultaneous multiplexed genome editing without any detectable random off-targets. In conclusion, our developed CGBE/ABE-PRSS system facilitates the selection of edited cells and holds promise in both basic engineering and gene therapy applications.

Puromycin reveals a distinct conformation of neuronal ribosomes.

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.

Protocol for assessing translation in living Drosophila imaginal discs by O-propargyl-puromycin incorporation.

Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging. We also provide details of quantification analysis. Significantly, OPP addition to methionine-containing media enables polypeptide labeling in living cells. Here, we study wing imaginal discs, an excellent model system for investigating growth, proliferation, pattern formation, differentiation, and cell death. For complete details on the use and execution of this protocol, please refer to Lee et al. (2018), Ji et al. (2019), and Kiparaki et al. (2022).1,2,3.

Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the Δorf2-8, Δorf2.4, and Δorf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the Δorf2-8 and Δorf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the Δorf2.4-puro replicon by inserting a puromycin-resistant gene into the Δorf2.4 replicon. We observed the more sustained replication of the Δorf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.

Large-scale analysis of mRNA sequences localized near the start and amber codons and their impact on the diversity of mRNA display libraries.

Extremely diverse libraries are essential for effectively selecting functional peptides or proteins, and mRNA display technology is a powerful tool for generating such libraries with over 1012-1013 diversity. Particularly, the protein-puromycin linker (PuL)/mRNA complex formation yield is determining for preparing the libraries. However, how mRNA sequences affect the complex formation yield remains unclear. To study the effects of N-terminal and C-terminal coding sequences on the complex formation yield, puromycin-attached mRNAs containing three random codons after the start codon (32768 sequences) or seven random bases next to the amber codon (6480 sequences) were translated. Enrichment scores were calculated by dividing the appearance rate of every sequence in protein-PuL/mRNA complexes by that in total mRNAs. The wide range of enrichment scores (0.09-2.10 for N-terminal and 0.30-4.23 for C-terminal coding sequences) indicated that the N-terminal and C-terminal coding sequences strongly affected the complex formation yield. Using C-terminal GGC-CGA-UAG-U sequences, which resulted in the highest enrichment scores, we constructed highly diverse libraries of monobodies and macrocyclic peptides. The present study provides insights into how mRNA sequences affect the protein/mRNA complex formation yield and will accelerate the identification of functional peptides and proteins involved in various biological processes and having therapeutic applications.

Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters.

CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.

Puromycin Prodrug Activation by Thioredoxin Reductase Overcomes Its Promiscuous Cytotoxicity.

Overexpression of the selenoprotein thioredoxin reductase (TrxR) has been documented in malignant tissues and is of pathological significance for many types of tumors. The antibiotic puromycin (Puro) is a protein synthesis inhibitor causing premature polypeptide chain termination during translation. The well-defined action mechanism of Puro makes it a useful tool in biomedical studies. However, the nonselective cytotoxicity of Puro limits its therapeutic applications. We report herein the construction and evaluation of two Puro prodrugs, that is, S1-Puro with a five-membered cyclic disulfide trigger and S2-Puro with a linear disulfide trigger. S1-Puro is selectively activated by TrxR and shows the TrxR-dependent cytotoxicity to cancer cells, while S2-Puro is readily activated by thiols. Furthermore, S1-Puro displays higher stability in plasma than S2-Puro. We expect that this prodrug strategy may promote the further development of Puro as a therapeutic agent.

Inherently Emissive Puromycin Analogues for Live Cell Labelling.

Puromycin derivatives containing an emissive thieno[3,4-d]-pyrimidine core, modified with azetidine and 3,3-difluoroazetidine as Me2 N surrogates, exhibit translation inhibition and bactericidal activity similar to the natural antibiotic. The analogues are capable of cellular puromycylation of nascent peptides, generating emissive products without any follow-up chemistry. The 3,3-difluoroazetidine-containing analogue is shown to fluorescently label newly translated peptides and be visualized in both live and fixed HEK293T cells and rat hippocampal neurons.

Minnelide Markedly Reduces Proteinuria in Mice with Adriamycin Nephropathy by Protecting Against Podocyte Injury.

Minimal change disease (MCD) is the most common cause of idiopathic nephrotic syndrome in children. The current major therapy is hormones for most steroid-sensitive patients. However, many patients have recurrent relapses of the disease and require long-term immunosuppression, leading to significant morbidity due to the side effects of the drugs. Therefore, better drugs need to be urgently explored to treat nephrotic syndrome while avoiding the side effects of drugs. Minnelide, a water-soluble prodrug of triptolide, has been proved to be effective in treating cancers in many clinical trials. This study aimed to investigate the therapeutic effect of minnelide in mice with adriamycin (ADR) nephropathy, its underlying protection mechanisms, and its reproductive toxicity. Minnelide was administered intraperitoneally to 6-8-week female mice with adriamycin nephropathy for 2 weeks, and the urine, blood, and kidney tissues were taken to analyze the therapeutic effect. In addition, we evaluated reproductive toxicity by measuring the levels of gonadal hormones and observing the histological changes in ovaries and testes. Primary mouse podocytes were exposed to puromycin (PAN) to damage the cytoskeleton and induce apoptosis, and then, triptolide was used to evaluate the therapeutic effect and underlying protection mechanisms in vitro. It was observed that minnelide dramatically alleviated proteinuria and apoptosis in mice with adriamycin nephropathy. In vitro, triptolide ameliorated puromycin-induced cytoskeletal rearrangement and apoptosis via reactive oxygen species-mediated mitochondrial pathway. In addition, minnelide caused no reproductive toxicity to male and female mice. The results suggested that minnelide might be a promising drug for nephrotic syndrome.

Optical Control of Translation with a Puromycin Photoswitch.

Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products have emerged that interfere with the ribosomal function, such as puromycin, which mimics an aminoacyl tRNA and causes premature chain termination. Here, we introduce a photoswitchable version of puromycin that, in effect, puts translation under optical control. Our compound, termed puroswitch, features a diazocine that allows for reversible and nearly quantitative isomerization and pharmacological modulation. Its synthesis involves a new photoswitchable amino acid building block. Puroswitch shows little activity in the dark and becomes substantially more active and cytotoxic, in a graded fashion, upon irradiation with various wavelengths of visible light. In vitro translation assays confirm that puroswitch inhibits translation with a mechanism similar to that of puromycin itself. Once incorporated into nascent proteins, puroswitch reacts with standard puromycin antibodies, which allows for tracking de novo protein synthesis using western blots and immunohistochemistry. As a cell-permeable small molecule, puroswitch can be used for nascent proteome profiling in a variety of cell types, including primary mouse neurons. We envision puroswitch as a useful biochemical tool for the optical control of translation and for monitoring newly synthesized proteins in defined locations and at precise time points.

Anti-ANGPTL3-FLD monoclonal antibody treatment ameliorates podocyte lesions through attenuating mitochondrial damage.

Proteinuria, an indication of kidney disease, is caused by the malfunction of podocytes, which play a key role in maintaining glomerular filtration. Angiopoietin-like 3 (ANGPTL3) has been documented to have a cell-autonomous involvement in podocytes, and deletion of Angptl3 in podocytes reduced proteinuria in adriamycin-induced nephropathy. Here, we developed a monoclonal antibody (mAb) against ANGPTL3 to investigate its effects on podocyte injury in an ADR nephropathy mouse model and puromycin (PAN) induced podocyte damage in vitro. The mAb against the human ANGPTL3-FLD sequence (5E5F6) inhibited the binding of ANGPTL3-FLD to integrin ß3. Treatment with the 5E5F6 mAb in ADR nephropathy mice mitigated proteinuria and led to a significant decline in podocyte apoptosis, reactive oxygen species (ROS) generation and mitochondrial fragmentation. In PAN-induced podocyte damage in vitro, the 5E5F6 mAb blocked the ANPGPLT3-mediated activation of integrin αvß3 and Rac1, which regulated the mitochondrial homeostasis. Altogether, anti-ANGPLT3-FLD mAb attenuates proteinuria and podocyte lesions in ADR mice models, as well as PAN-induced podocyte damage, in part through regulating mitochondrial functions. Our study provides a therapeutic approach for targeting ANGPTL3 in proteinuric kidney disease.