Pharmacological induction of translational readthrough of nonsense mutations in the retinoblastoma (RB1) gene.

The retinoblastoma protein (Rb) is encoded by the RB1 tumor suppressor gene. Inactivation of RB1 by inherited or somatic mutation occurs in retinoblastoma and various other types of tumors. A significant fraction (25.9%) of somatic RB1 mutations are nonsense substitutions leading to a premature termination codon (PTC) in the RB1 coding sequence and expression of truncated inactive Rb protein. Here we show that aminoglycoside G418, a known translational readthrough inducer, can induce full-length Rb protein in SW1783 astrocytoma cells with endogenous R579X nonsense mutant RB1 as well as in MDA-MB-436 breast carcinoma cells transiently transfected with R251X, R320X, R579X or Q702X nonsense mutant RB1 cDNA. Readthrough was associated with increased RB1 mRNA levels in nonsense mutant RB1 cells. Induction of full-length Rb protein was potentiated by the cereblon E3 ligase modulator CC-90009. These results suggest that pharmacological induction of translational readthrough could be a feasible strategy for therapeutic targeting of tumors with nonsense mutant RB1.

Novel compounds that synergize with aminoglycoside G418 or eRF3 degraders for translational readthrough of nonsense mutant TP53 and PTEN.

The TP53 and PTEN tumour suppressor genes are inactivated by nonsense mutations in a significant fraction of human tumours. TP53 nonsense mutatant tumours account for approximately one million new cancer cases per year worldwide. We have screened chemical libraries with the aim of identifying compounds that induce translational readthrough and expression of full-length p53 protein in cells with nonsense mutation in this gene. Here we describe two novel compounds with readthrough activity, either alone or in combination with other known readthrough-promoting substances. Both compounds induced levels of full-length p53 in cells carrying R213X nonsense mutant TP53. Compound C47 showed synergy with the aminoglycoside antibiotic and known readthrough inducer G418, whereas compound C61 synergized with eukaryotic release factor 3 (eRF3) degraders CC-885 and CC-90009. C47 alone showed potent induction of full-length PTEN protein in cells with different PTEN nonsense mutations. These results may facilitate further development of novel targeted cancer therapy by pharmacological induction of translational readthrough.

Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine.

TP53 nonsense mutations in cancer produce truncated inactive p53 protein. We show that 5-FU metabolite 5-Fluorouridine (FUr) induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53. Ribosome profiling visualized translational readthrough at the R213X premature stop codon and demonstrated that FUr-induced readthrough is less permissive for canonical stop codon readthrough compared to aminoglycoside G418. FUr is incorporated into mRNA and can potentially base-pair with guanine, allowing insertion of Arg tRNA at the TP53 R213X UGA premature stop codon and translation of full-length wild-type p53. We confirmed that full-length p53 rescued by FUr triggers tumor cell death by apoptosis. FUr also restored full-length p53 in TP53 R213X mutant human tumor xenografts in vivo. Thus, we demonstrate a novel strategy for therapeutic rescue of nonsense mutant TP53 and suggest that FUr should be explored for treatment of patients with TP53 nonsense mutant tumors.

Ipidacrine (Axamon), A Reversible Cholinesterase Inhibitor, Improves Erectile Function in Male Rats With Diabetes Mellitus-Induced Erectile Dysfunction.

BACKGROUND: Management of diabetes mellitus-induced erectile dysfunction (DMED) is challenging because of its insufficient responses to phosphodiesterase type 5 inhibitors. AIM: To compare the effects of ipidacrine, a reversible cholinesterase inhibitor, and sildenafil on DMED in a rat model of streptozotocin (STZ)-induced diabetes. METHODS: Erectile dysfunction (ED) caused by STZ-induced diabetes mellitus was modeled in adult male Wistar rats, which were randomized to 4 groups: untreated diabetic rats, sildenafil (5 mg/kg), ipidacrine (3.6 mg/kg) and ipidacrine (6.7 mg/kg). The test drug (ipidacrine), comparator (sildenafil) or control substance (1% starch solution) were administered orally for 5 days or 14 days. Erectile function was assessed by the change in the maximum intracavernous pressure (ICPmax) following cavernous nerve electrical stimulation. The mean arterial pressure (MAP) was recorded, and the ICPmax/MAP ratio was calculated. Sexual behavior, cholinesterase activity and blood testosterone level tests assessed. MAIN OUTCOME MEASURE: The quantitative value of ICPmax/MAP 14 days after the start of administration of the test drug and the comparison drug. RESULTS: Animals with STZ-induced diabetes mellitus showed a significant decrease in ICPmax and ICPmax/MAP ratio compared to the intact control group. When ipidacrine was administered to rats with DMED for 14 days, an increase in these indicators was noted. It was proved that ipidacrine at a dose of 6.7 mg/kg has noninferiority compared to sildenafil on the DMED model. Significant increase in ICPmax compared to STZ-control after electrostimulation of the cavernous nerve was recorded following administration of ipidacrine at a dose of 6.7 mg/kg (P < .05) and sildenafil at a dose 5 mg/kg (P < .05). Neither the test drug, nor the comparator were associated with increase in testosterone levels in blood; as well both drugs did not promote activation of sexual behavior. CLINICAL IMPLICATIONS: Ipidacrine may be considered as an effective therapy for DMED but needs to be verified in human investigations. STRENGTHS & LIMITATIONS: The role of ipidacrine, was firstly demonstrated in rats with DMED. However, the results were obtained in animal experiments, and will be further tested in the study of receptor interactions and the determination of cellular targets. CONCLUSION: This is the first study to show that administration of ipidacrine, the reversible cholinesterase inhibitor, improved erectile function in diabetic rats and these results may be beneficial in further studies using ipidacrine for treatment of DMED, particularly in non-responders to PDE5 inhibitors. Bykov V, Gushchina E, Morozov S, et al. Ipidacrine (Axamon), A Reversible Cholinesterase Inhibitor, Improves Erectile Function in Male Rats With Diabetes Mellitus-Induced Erectile Dysfunction. Sex Med 2022;10:100477.

Optimization of application schedule of camphecene, a novel anti-influenza compound, based on its pharmacokinetic characteristics.

Due to high variability and rapid life cycle, influenza virus is able to develop drug resistance against direct-acting antivirals. Development of novel virus-in113039hibiting drugs is therefore important goal. Previously, we identified camphor derivative, camphecene, as an effective anti-influenza compound. In the present study, we optimize the regimen of its application to avoid high sub-toxic concentrations. The protective activity of camphecene was assessed on the model of lethal pneumonia of mice caused by influenza viruses. Camphecene was administered either once a day or four times a day, alone or in combination with Tamiflu. Mortality and viral titer in the lungs were studied. Pharmacokinetics of camphecene was studied in rabbits. We have demonstrated that camphecene, being used every 6 h at a dose of 7.5 mg/kg/day, results in antiviral effect that was statistically equal to the effect of 100 mg/kg/day once a day, that is, the same effect was achieved by 13 times lower daily dose of the drug. This effect was manifested in decrease of mortality and decrease of virus' titer in the lungs. The studies of pharmacokinetics of camphecene have demonstrated that it does not accumulate in blood plasma and that its m ultiple applications with dosage interval of 65 min are safe. In addition, the results of the study demonstrate also that camphecene possesses additive effect with Tamiflu, allowing to decrease the dose of the latter. The results suggest that due to safety and efficacy, camphecene can be further developed as potential anti-influenza remedy.

Quantitative determination of new anti-inflammatory drug indomenthyl and its metabolite in rabbit plasma by HPLC-MS/MS.

Background: Indomenthyl is an innovative anti-inflammatory drug with a high analgesic activity. Indomenthyl releases indomethacin under the influence of neutrophil esterases in the inflammation focus. Methodology/results: This research is aimed at developing a highly sensitive method for the quantitative determination of indomenthyl and its active metabolite indomethacin in rabbit plasma by HPLC-MS/MS. Protein precipitation and extraction with acetonitrile were used for analyte isolation from plasma according to the QuEChERS principle. The target quantitative ion pairs m/z were respectively 496.4 → 358.0 for indomenthyl, 358.0 → 139.5 for indomethacin, and 340.1 → 202.1 for the IS. Conclusion: The calibration curve was linear over the range 0.1-1000 ng mL-1. The technique was applied to the pharmacokinetic study at a dose of 25 mg kg-1 to rabbits.

Assessing the natural and anthropogenic radionuclide activities of the Pechora River estuary: Bottom sediments and water (Arctic Ocean Basin).

This paper studies the activity of natural and technogenic radionuclides in bottom sediments and surface water of the Pechora River estuary, which is the largest Arctic river within the European part of Russia. The relevance of conducting radioecological studies of this region is associated with active oil and gas activities in the Pechora basin and the presence of potential sources of radiation hazard. The average activities of 137Cs, 226Ra, 232Th, and 40K in bottom sediments were 0.16, 18.72, 13.36, and 373.59 Bq·kg-1, respectively. The average activity of 226Ra in the bottom sediments of the Pechora delta was two times higher than in other rivers of the Arctic basin of the European part of Russia, which is associated with oil and gas activities that are carried out in the Pechora basin. This is due to the fact that the decay products of uranium are the main by-products of oil and gas production. The specific activity of technogenic 137Cs in bottom sediments of the Pechora River delta was low and did not exceed 2.2 Bq·kg-1. We did not detect any radioactive 90Sr in bottom sediments. The low 137Cs activity in the bottom sediments of the Pechora delta was associated with both the larger granulometric composition of the sediments in comparison with the estuaries of the Siberian rivers, and with low concentrations of 137Cs in the terrigenous material (soil) entering the river from the catchment area. This was due to the low levels of 137Cs contamination in the soil of the Pechora river basin. We did not find any negative radiation effects of the underground nuclear explosion "Pyrite" carried out in the northern part of the delta, since there were no other technogenic radionuclides. The values of the radionuclides 3H, 137Cs, 226Ra, 232Th and 40K, as well as the total alpha activity in the surface waters of the Pechora River delta, turned out to be below the detection limit. In surface waters, we were able to determine only the total beta activity, which varied over a range from 19.6 to 59.6 Bq·m-3 with an average value of 33.9 Bq·m-3, which was significantly lower than the radiation safety standards. The values of the radiation hazard parameters were below the world average levels and were not significantly hazardous to the health of people living in the area of the Pechora River delta. We concluded that at present, in the Pechora River delta, there are pastures for reindeer with low levels of natural and man-made radioactivity. These conducted studies are the source material in a series of comprehensive studies of the current radioecological state of the tundra territories of the European part of Russia.

Mutant p53-reactivating compound APR-246 synergizes with asparaginase in inducing growth suppression in acute lymphoblastic leukemia cells.

Asparaginase depletes extracellular asparagine in the blood and is an important treatment for acute lymphoblastic leukemia (ALL) due to asparagine auxotrophy of ALL blasts. Unfortunately, resistance occurs and has been linked to expression of the enzyme asparagine synthetase (ASNS), which generates asparagine from intracellular sources. Although TP53 is the most frequently mutated gene in cancer overall, TP53 mutations are rare in ALL. However, TP53 mutation is associated with poor therapy response and occurs at higher frequency in relapsed ALL. The mutant p53-reactivating compound APR-246 (Eprenetapopt/PRIMA-1Met) is currently being tested in phase II and III clinical trials in several hematological malignancies with mutant TP53. Here we present CEllular Thermal Shift Assay (CETSA) data indicating that ASNS is a direct or indirect target of APR-246 via the active product methylene quinuclidinone (MQ). Furthermore, combination treatment with asparaginase and APR-246 resulted in synergistic growth suppression in ALL cell lines. Our results thus suggest a potential novel treatment strategy for ALL.

A thiol-bound drug reservoir enhances APR-246-induced mutant p53 tumor cell death.

The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. The compound APR-246 (PRIMA-1Met/Eprenetapopt) is converted to methylene quinuclidinone (MQ) that targets mutant p53 protein and perturbs cellular antioxidant balance. APR-246 is currently tested in a phase III clinical trial in myelodysplastic syndrome (MDS). By in vitro, ex vivo, and in vivo models, we show that combined treatment with APR-246 and inhibitors of efflux pump MRP1/ABCC1 results in synergistic tumor cell death, which is more pronounced in TP53 mutant cells. This is associated with altered cellular thiol status and increased intracellular glutathione-conjugated MQ (GS-MQ). Due to the reversibility of MQ conjugation, GS-MQ forms an intracellular drug reservoir that increases availability of MQ for targeting mutant p53. Our study shows that redox homeostasis is a critical determinant of the response to mutant p53-targeted cancer therapy.

Correction: APR-246 reactivates mutant p53 by targeting cysteines 124 and 277.

Since publication of this article, the authors have noticed that there was an error in Fig. 1d, third panel from left, "R273H + 200 µM MQ-H" should be "R273H + 200 µM MQ". Our corrections do not affect the original conclusions of this paper.

p53 as a hub in cellular redox regulation and therapeutic target in cancer.

The TP53 tumor suppressor gene encodes a DNA-binding transcription factor that regulates multiple cellular processes including cell growth and cell death. The ability of p53 to bind to DNA and activate transcription is tightly regulated by post-translational modifications and is dependent on a reducing cellular environment. Some p53 transcriptional target genes are involved in regulation of the cellular redox homeostasis, e.g. TIGAR and GLS2. A large fraction of human tumors carry TP53 mutations, most commonly missense mutations that lead to single amino acid substitutions in the core domain. Mutant p53 proteins can acquire so called gain-of-function activities and influence the cellular redox balance in various ways, for instance by binding of the Nrf2 transcription factor, a major regulator of cellular redox state. The DNA-binding core domain of p53 has 10 cysteine residues, three of which participate in holding a zinc atom that is critical for p53 structure and function. Several novel compounds that refold and reactivate missense mutant p53 bind to specific p53 cysteine residues. These compounds can also react with other thiols and target components of the cellular redox system, such as glutathione. Dual targeting of mutant p53 and redox homeostasis may allow more efficient treatment of cancer.

The Mutant p53-Targeting Compound APR-246 Induces ROS-Modulating Genes in Breast Cancer Cells.

TP53 is the most frequently mutated gene in human cancer and thus an attractive target for novel cancer therapy. Several compounds that can reactive mutant p53 protein have been identified. APR-246 is currently being tested in a phase II clinical trial in high-grade serous ovarian cancer. We have used RNA-seq analysis to study the effects of APR-246 on gene expression in human breast cancer cell lines. Although the effect of APR-246 on gene expression was largely cell line dependent, six genes were upregulated across all three cell lines studied, i.e., TRIM16, SLC7A11, TXNRD1, SRXN1, LOC344887, and SLC7A11-AS1. We did not detect upregulation of canonical p53 target genes such as CDKN1A (p21), 14-3-3σ, BBC3 (PUMA), and PMAIP1 (NOXA) by RNA-seq, but these genes were induced according to analysis by qPCR. Gene ontology analysis showed that APR-246 induced changes in pathways such as response to oxidative stress, gene expression, cell proliferation, response to nitrosative stress, and the glutathione biosynthesis process. Our results are consistent with the dual action of APR-246, i.e., reactivation of mutant p53 and modulation of redox activity. SLC7A11, TRIM16, TXNRD1, and SRXN1 are potential new pharmacodynamic biomarkers for assessing the response to APR-246 in both preclinical and clinical studies.

Inhibition of the glutaredoxin and thioredoxin systems and ribonucleotide reductase by mutant p53-targeting compound APR-246.

The tumor suppressor p53 is commonly inactivated in human tumors, allowing evasion of p53-dependent apoptosis and tumor progression. The small molecule APR-246 (PRIMA-1Met) can reactive mutant p53 in tumor cells and trigger cell death by apoptosis. The thioredoxin (Trx) and glutaredoxin (Grx) systems are important as antioxidants for maintaining cellular redox balance and providing electrons for thiol-dependent reactions like those catalyzed by ribonucleotide reductase and peroxiredoxins (Prxs). We show here that the Michael acceptor methylene quinuclidinone (MQ), the active form of APR-246, is a potent direct inhibitor of Trx1 and Grx1 by reacting with sulfhydryl groups in the enzymes. The inhibition of Trx1 and Grx1 by APR-246/MQ is reversible and the inhibitory efficiency is dependent on the presence of glutathione. APR-246/MQ also inhibits Trxs in mutant p53-expressing Saos-2 His-273 cells, showing modification of Trx1 and mitochondrial Trx2. Inhibition of the Trx and Grx systems leads to insufficient reducing power to deoxyribonucleotide production for DNA replication and repair and peroxiredoxin for removal of ROS. We also demonstrate that APR-246 and MQ inhibit ribonucleotide reductase (RNR) in vitro and in living cells. Our results suggest that APR-246 induces tumor cell death through both reactivations of mutant p53 and inhibition of cellular thiol-dependent redox systems, providing a novel strategy for cancer therapy.

Needling With 5-Fluorouracil (5-FU) After XEN Gel Stent Implantation: 6-Month Outcomes.

PURPOSE: The purpose of this study was to evaluate frequency, safety, and efficacy of needling in patients that underwent XEN Gel Stent implantation. METHODS: Retrospective case review of 19 eyes of 57 consecutive patients (61 eyes) with primary open-angle glaucoma or pseudoexfoliative glaucoma that previously underwent implantation of XEN45 alone or in combination with cataract surgery followed by needling procedure with 5-FU. Success was defined at 2 IOP levels: ≤21 mm Hg and ≤15 mm Hg, with or without additional glaucoma medications. Treatment failure was defined as IOP>21 mm Hg or <5 mm Hg, need for additional glaucoma surgery or loss of light perception. RESULTS: Totally 19 of 61 eyes that underwent XEN gel implantation had subsequent needling and were included. Preneedling IOP was 26.2±9.5 and postneedling IOP at last follow-up 15.4±3.7 mm Hg (P=0.0001). Overall success rates of 17 (90%) and 13 eyes (69%) were observed at the ≤21 mm Hg and ≤15 mm Hg level, respectively. Preneedling and postneedling visual acuity and number of medications remained unchanged (P>0.05). Two eyes (10%) were categorized as treatment failures. No major complications occurred. Mean follow-up was 203.8±142.2 (range, 22 to 456) days. CONCLUSIONS: Needling revision following XEN gel stent implantation showed a good IOP-lowering effect without significant increase in number of antiglaucoma medications, decrease in visual acuity, nor any major complications. Further studies with long-term follow-up and a larger number of patients are needed to fully assess the safety and efficacy of this procedure.

Role of Thiol Reactivity for Targeting Mutant p53.

Reactivation of mutant p53 has emerged as a promising approach for cancer therapy. Recent studies have identified several mutant p53-reactivating compounds that target thiol groups in mutant p53. Here we have investigated the relationship between thiol reactivity, p53 thermostabilization, mutant p53 refolding, mutant p53-dependent growth suppression, and induction of cell death. Analysis of the National Cancer Institute database revealed that Michael acceptors show the highest selectivity for mutant p53-expressing cells among analyzed thiol-reactive compounds. Further experimental testing demonstrated that Michael acceptors, aldehydes, imines, and primary alcohols can promote thermodynamic stabilization of mutant p53. Moreover, mild thiol reactivity, often coupled with combined chemical functional groups, such as in imines, aldehydes, and primary alcohols, can stimulate mutant p53 refolding. However, strong electrophile activity was associated with cellular toxicity. Our findings may open possibilities for rational design of novel potent and selective mutant p53-reactivating compounds.

APR-246 reactivates mutant p53 by targeting cysteines 124 and 277.

The TP53 tumor suppressor gene is frequently inactivated in human tumors by missense mutations in the DNA binding domain. TP53 mutations lead to protein unfolding, decreased thermostability and loss of DNA binding and transcription factor function. Pharmacological targeting of mutant p53 to restore its tumor suppressor function is a promising strategy for cancer therapy. The mutant p53 reactivating compound APR-246 (PRIMA-1Met) has been successfully tested in a phase I/IIa clinical trial. APR-246 is converted to the reactive electrophile methylene quinuclidinone (MQ), which binds covalently to p53 core domain. We identified cysteine 277 as a prime binding target for MQ in p53. Cys277 is also essential for MQ-mediated thermostabilization of wild-type, R175H and R273H mutant p53, while both Cys124 and Cys277 are required for APR-246-mediated functional restoration of R175H mutant p53 in living tumor cells. These findings may open opportunities for rational design of novel mutant p53-targeting compounds.

Radioprotective and radiomitigative effects of BP-C2, a novel lignin-derived polyphenolic composition with ammonium molybdate, in two mouse strains exposed to total body irradiation.

PURPOSE: There remains an unmet medical need for radioprotective and mitigative agents. BP-C2 is a novel lignin-derived polyphenolic composition with ammonium molybdate, developed as radioprotector/radiomitigator. OBJECTIVES: The present study evaluated BP-C2 for the mitigation of acute radiation syndrome (ARS). METHODS: A total-body irradiation mouse model (TBI, 4.0-8.0 Gy) was used in the study. RESULTS: In a 30-day survival study, performed in CBA mice, BP-C2, at a dosage of 81.0 mg/kg, improved survival (dose reduction factor (DRF) = 1.1) and increased the formation of endogenous spleen colony-forming units (CFU). In C57BL/6 mice, BP-C2, when administered daily for 7 days, starting 24 hours after TBI, also improved survival. In animals irradiated with 5.0 Gy, BP-C2 increased the number of CFUs (6.7 ± 5.1) compared to the 5.0 Gy placebo group (2.3 ± 2.3, p = .0245). The number of surviving intestinal crypts was maintained in the 5.0 Gy BP-C2 group (133.7 ± 13.9), in contrast to the 5.0 Gy placebo group (124.2 ± 10.5, p < .0023). BP-C2 also increased the number of LGR5 + positive cells in intestinal crypts. CONCLUSION: BP-C2 mitigates radiation-induced damage in mid-lethal range of radiation doses. Effects are mediated by enhancement of extramedullar hematopoiesis in the spleen and a protective effect on the intestinal epithelium.

Targeting mutant p53 for efficient cancer therapy.

The tumour suppressor gene TP53 is the most frequently mutated gene in cancer. Wild-type p53 can suppress tumour development by multiple pathways. However, mutation of TP53 and the resultant inactivation of p53 allow evasion of tumour cell death and rapid tumour progression. The high frequency of TP53 mutation in tumours has prompted efforts to restore normal function of mutant p53 and thereby trigger tumour cell death and tumour elimination. Small molecules that can reactivate missense-mutant p53 protein have been identified by different strategies, and two compounds are being tested in clinical trials. Novel approaches for targeting TP53 nonsense mutations are also underway. This Review discusses recent progress in pharmacological reactivation of mutant p53 and highlights problems and promises with these strategies.