Biomarkers and Novel Therapies of Diabetic Neuropathy: An Updated Review.

Diabetic neuropathy is a persistent consequence of the biochemical condition known as diabetes mellitus. As of now, the identification and management of diabetic neuropathy continue to be problematic due to problems related to the safety and efficacy of existing therapies. This study examines biomarkers, molecular and cellular events associated with the advancement of diabetic neuropathy, as well as the existing pharmacological and non-pharmacological treatments employed. Furthermore, a holistic and mechanism-centric drug repurposing approach, antioxidant therapy, Gene and Cell therapies, Capsaicin and other spinal cord stimulators and lifestyle interventions are pursued for the identification, treatment and management of diabetic neuropathy. An extensive literature survey was done on databases like PubMed, Elsevier, Science Direct and Springer using the keywords "Diabetic Neuropathy", "Biomarkers", "Cellular and Molecular Mechanisms", and "Novel Therapeutic Targets".Thus, we may conclude that non-pharmacological therapies along with palliative treatment, may prove to be crucial in halting the onset of neuropathic symptoms and in lessening those symptoms once they have occurred.

Repurposing prescribed hydromorphone: Alternative uses of safer supply and tablet-injectable opioid agonist treatment to meet unaddressed health needs.

BACKGROUND: In response to the ongoing overdose crisis in Canada, a number of opioid agonist treatment and safer supply programs provide people at high overdose risk with daily-dispensed tablet hydromorphone, with some requiring witnessed ingestion and others providing take-away doses. While these programs are intended to reduce overdose events by limiting people's use of the contaminated drug supply, the experiences of people receiving hydromorphone vary. In this article we explore the ways people repurpose hydromorphone to address unmet needs. METHODS: This article draws on in-depth qualitative interviews from two studies evaluating hydromorphone tablet distribution programs in British Columbia, Canada. We used thematic analysis to identify themes related to repurposing hydromorphone. We compared themes across the two studies to identify any similarities or differences in relation to the ways study participants discussed repurposing hydromorphone tablets. We utilize vignettes - snapshots of participant experiences - to analyse and represent the data. RESULTS: Four vignettes demonstrate how hydromorphone tablets are often being used to address and resolve unmet needs of people who use drugs. While most participants reported reducing their use of illicit drugs, a variety of instrumental uses of tablet hydromorphone were also discussed, including reducing anxiety, addressing sleep issues, withdrawal management, and managing chronic pain. CONCLUSION: Our findings demonstrate how people who use drugs are maximizing the benefits of tablet hydromorphone distribution to address unmet needs. Hydromorphone distribution programs represent a public health and harm reduction intervention that is usefully addressing experiences related to structural vulnerabilities (such as inadequate pain management), which are often overlooked amongst stigmatized groups.

A deep drug prediction framework for viral infectious diseases using an optimizer-based ensemble of convolutional neural network: COVID-19 as a case study.

The SARS-CoV-2 outbreak highlights the persistent vulnerability of humanity to epidemics and emerging microbial threats, emphasizing the lack of time to develop disease-specific treatments. Therefore, it appears beneficial to utilize existing resources and therapies. Computational drug repositioning is an effective strategy that redirects authorized drugs to new therapeutic purposes. This strategy holds significant promise for newly emerging diseases, as drug discovery is a lengthy and expensive process. Through this study, we present an ensemble method based on the convolutional neural network integrated with genetic algorithm and deep forest classifier for virus-drug association prediction (CGDVDA). We generated feature vectors by combining drug chemical structure and virus genomic sequence-based similarities, and extracted prominent deep features by applying the convolutional neural network. The convoluted features are optimized using the genetic algorithm and classified using the ensemble deep forest classifier to predict novel virus-drug associations. The proposed method predicts drugs for COVID-19 and other viral diseases in the dataset. The model could achieve ROC-AUC scores of 0.9159 on fivefold cross-validation. We compared the performance of the model with state-of-the-art approaches and classifiers. The experimental results and case studies illustrate the efficacy of CGDVDA in predicting drugs against viral infectious diseases.

Findings from the individualized management of a patient with Acyl-CoA Oxidase-1 (ACOX1) deficiency: A bedside-to-bench-to-bedside strategy.

Acyl-CoA Oxidase-1 (ACOX1) deficiency (MIM 264470) is an autosomal recessive disease characterized by impairments in the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, which is the first step in the catalysis of the ß-oxidative breakdown of very long chain fatty acids (VLCFA) occuring in peroxisomes. The deleterious accumulation of VLCFA in several organs, including the brain, is a key biochemical feature of this disease which has devastating neurological consequences. ACOX1 deficiency is ultra-rare; as such, few studies have been conducted to determine the leading causes of symptoms or uncover new therapeutics. When confronted with one such case, we decided to bring drug discovery tools to the patient's bedside in an attempt to identify a cure. A skin biopsy was performed on a young patient with ACOX1 deficiency, following which screening technologies and mass spectrometry analysis techniques were applied to design a cellular assay that enabled the direct measurement of the effect of small molecules on the patient's primary fibroblasts. This approach is particularly well adapted to inherited metabolic disorders such as ACOX1 deficiency. Through the evaluation of a proprietary library of repurposable drugs, we found that the anthelmintic drug niclosamide led to a significant reduction in VLCFA in vitro. This drug was subsequently administered to the patient for more than six years. This study outlines the screening and drug selection processes. Additionally, we present our comprehensive clinical and biochemical findings that aided in understanding the patient's natural history and analysis of the progression of the patient's symptoms throughout the treatment period. Although the patient's overall lifespan was extended compared to the average age at death in severe early onset cases of ACOX1 deficiency, we did not observe any definitive evidence of clinical or biochemical improvement during niclosamide treatment. Nonetheless, our study shows a good safety profile of long-term niclosamide administration in a child with a rare neurodegenerative disease, and illustrates the potential of individualized therapeutic strategies in the management of inherited metabolic disorders, which could benefit both patients and the broader scientific and medical communities.

Beyond chloroquine: Cationic amphiphilic drugs as endosomal escape enhancers for nucleic acid therapeutics.

Nucleic acid (NA) therapeutics have the potential to treat or prevent a myriad of diseases but generally require cytosolic delivery to be functional. NA drugs are therefore often encapsulated into delivery systems that mediate effective endocytic uptake by target cells, but unfortunately often display limited endosomal escape efficiency. This review will focus on the potential of repurposing cationic amphiphilic drugs (CADs) to enhance endosomal escape. In general terms, CADs are small molecules with one or more hydrophobic groups and a polar domain containing a basic amine. CADs have been reported to accumulate in acidified intracellular compartments (e.g., endosomes and lysosomes), integrate in cellular membranes and alter endosomal trafficking pathways, ultimately resulting in improved cytosolic release of the endocytosed cargo. As many CADs are widely used drugs, their repurposing offers opportunities for combination therapies with NAs.

Drug repositioning identifies potential autophagy inhibitors for the LIR motif p62/SQSTM1 protein.

Autophagy is a critical cellular process for degrading damaged organelles and proteins under stressful conditions and has casually been shown to contribute to tumor survival and drug resistance. Sequestosome-1 (SQSTM1/p62) is an autophagy receptor that interacts with its binding partners via the LC3-interacting region (LIR). The p62 protein has been a highly researched target for its critical role in selective autophagy. In this study, we aimed to identify FDA-approved drugs that bind to the LIR motif of p62 and inhibit its LIR function, which could be useful targets for modulating autophagy. To this, the homology model of the p62 protein was predicted using biological data, and docking analysis was performed using Molegro Virtual Docker and PyRx softwares. We further assessed the toxicity profile of the drugs using the ProTox-II server and performed dynamics simulations on the effective candidate drugs identified. The results revealed that the kanamycin, velpatasvir, verteporfin, and temoporfin significantly decreased the binding of LIR to the p62 protein. Finally, we experimentally confirmed that Kanamycin can inhibit autophagy-associated acidic vesicular formation in breast cancer MCF-7 and MDA-MB 231 cells. These repositioned drugs may represent novel autophagy modulators in clinical management, warranting further investigation.

Trifluoperazine effect on human sperm: the accumulation of reactive oxygen species and the decrease in the mitochondrial membrane potential.

A strong link between antipsychotic drug use and reduced human sperm quality has been reported. Trifluoperazine (TFP), a commonly used antipsychotic, is now being explored for anticancer applications. Although there are hints that TFP might affect the male reproductive system, its impact on human sperm quality remains uncertain. Using a human sperm and TFP in vitro coculture system, we examined the effect of TFP (12.5, 25, 50 and 100µM) on human sperm function and physiological parameters. The results showed that 50µM and 100µM TFP induced the accumulation of reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential (MMP) of human sperm, leading to decreased sperm viability, while 25µM TFP inhibited only the penetration ability, total sperm motility, and progressive motility. Although 12.5µM and 25µM TFP increased [Ca2+]i in human sperm, they did not affect capacitation or the acrosome reaction. These results may be explained by the observation that 12.5µM and 25µM TFP did not increase tyrosine phosphorylation in human sperm, although TFP increased [Ca2+]i in a time-course traces similar to that of progesterone. Our results indicated that TFP could cause male reproductive toxicity by inducing the accumulation of ROS and a decrease in the MMP in human sperm.

Exploring Niclosamide as a Multi-target Drug Against SARS-CoV-2: Molecular Dynamics Simulation Studies on Host and Viral Proteins.

Niclosamide has emerged as a promising repurposed drug against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In vitro studies suggested that niclosamide inhibits the host transmembrane protein 16F (hTMEM16F), crucial for lipid scramblase activity, which consequently reduces syncytia formation that aids viral spread. Based on other in vitro reports, niclosamide may also target viral proteases such as papain-like protease (PLpro) and main protease (Mpro), essential for viral replication and maturation. However, the precise interactions by which niclosamide interacts with these multiple targets remain largely unclear. Docking and molecular dynamics (MD) simulation studies were undertaken based on a homology model of the hTMEM16F and available crystal structures of SARS-CoV-2 PLpro and Mpro. Niclosamide was observed to bind stably throughout a 400 ns MD simulation at the extracellular exit gate of the hTMEM16F tunnel, forming crucial interactions with residues spanning the TM1-TM2 loop (Gln350), TM3 (Phe481), and TM5-TM6 loop (Lys573, Glu594, and Asp596). Among the SARS-CoV-2 proteases, niclosamide was found to interact effectively with conserved active site residues of PLpro (Tyr268), exhibiting better stability in comparison to the control inhibitor, GRL0617. In conclusion, our in silico analyses support niclosamide as a multi-targeted drug inhibiting viral and host proteins involved in SARS-CoV-2 infections.

Exploring the anticancer mechanism of cardiac glycosides using proteome integral solubility alteration approach.

BACKGROUND AND AIMS: Cardiac glycosides (CGs), traditionally used for heart failure, have shown potential as anti-cancer agents. This study aims to explore their multifaceted mechanisms in cancer cell biology using proteome integral solubility alteration (PISA), focusing on the interaction with key proteins implicated in cellular metabolism and mitochondrial function. METHODS: We conducted lysate-based and intact-cell PISA assays on cancer cells treated with CGs (Digoxin, Digitoxin, Ouabain) to analyze protein solubility changes. This was followed by mass spectrometric analysis and bioinformatics to identify differentially soluble proteins (DSPs). Molecular docking simulations were performed to predict protein-CG interactions. Public data including gene expression changes upon CG treatment were re-analyzed for validation. RESULTS: The PISA assays revealed CGs' broad-spectrum interactions, particularly affecting proteins like PKM2, ANXA2, SLC16A1, GOT2 and GLUD1. Molecular docking confirmed stable interactions between CGs and these DSPs. Re-analysis of public data supported the impact of CGs on cancer metabolism and cell signaling pathways. CONCLUSION: Our findings suggest that CGs could be repurposed for cancer therapy by modulating cellular processes. The PISA data provide insights into the polypharmacological effects of CGs, warranting further exploration of their mechanisms and clinical potential.

Anticancer role of flubendazole: Effects and molecular mechanisms (Review).

Flubendazole, an anthelmintic agent with a well-established safety profile, has emerged as a promising anticancer drug that has demonstrated efficacy against a spectrum of cancer types over the past decade. Its anticancer properties encompass a multifaceted mechanism of action, including the inhibition of cancer cell proliferation, disruption of microtubule dynamics, regulation of cell cycle, autophagy, apoptosis, suppression of cancer stem cell characteristics, promotion of ferroptosis and inhibition of angiogenesis. The present review aimed to provide a comprehensive overview of the molecular underpinnings of the anticancer activity of flubendazole, highlighting key molecules and regulatory pathways. Given the breadth of the potential of flubendazole, further research is imperative to identify additional cancer types sensitive to flubendazole, refine experimental methodologies for enhancing its reliability, uncover synergistic drug combinations, improve its bioavailability and explore innovative administration methods. The present review provided a foundation for future studies on the role of flubendazole in oncology and described its molecular mechanisms of action.

Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria.

While highly effective at killing Gram-positive bacteria, auranofin lacks significant activity against Gram-negative species for reasons that largely remain unclear. Here, we aimed to elucidate the molecular mechanisms underlying the low susceptibility of the Gram-negative model organism Escherichia coli to auranofin when compared to the Gram-positive model organism Bacillus subtilis. The proteome response of E. coli exposed to auranofin suggests a combination of inactivation of thiol-containing enzymes and the induction of systemic oxidative stress. Susceptibility tests in E. coli mutants lacking proteins upregulated upon auranofin treatment suggested that none of them are directly involved in E. coli's high tolerance to auranofin. E. coli cells lacking the efflux pump component TolC were more sensitive to auranofin treatment, but not to an extent that would fully explain the observed difference in susceptibility of Gram-positive and Gram-negative organisms. We thus tested whether E. coli's thioredoxin reductase (TrxB) is inherently less sensitive to auranofin than TrxB from B. subtilis, which was not the case. However, E. coli strains lacking the low-molecular-weight thiol glutathione, but not glutathione reductase, showed a high susceptibility to auranofin. Bacterial cells expressing the genetically encoded redox probe roGFP2 allowed us to observe the oxidation of cellular protein thiols in situ. Based on our findings, we hypothesize that auranofin leads to a global disturbance in the cellular thiol redox homeostasis in bacteria, but Gram-negative bacteria are inherently more resistant due to the presence of drug export systems and high cellular concentrations of glutathione.IMPORTANCEAuranofin is an FDA-approved drug for the treatment of rheumatoid arthritis. However, it has also high antibacterial activity, in particular against Gram-positive organisms. In the current antibiotics crisis, this would make it an ideal candidate for drug repurposing. However, its much lower activity against Gram-negative organisms prevents its broad-spectrum application. Here we show that, on the level of the presumed target, there is no difference in susceptibility between Gram-negative and Gram-positive species: thioredoxin reductases from both Escherichia coli and Bacillus subtilis are equally inhibited by auranofin. In both species, auranofin treatment leads to oxidative protein modification on a systemic level, as monitored by proteomics and the genetically encoded redox probe roGFP2. The single largest contributor to E. coli's relative resistance to auranofin seems to be the low-molecular-weight thiol glutathione, which is absent in B. subtilis and other Gram-positive species.

Bioinformatics Approaches in the Development of Antifungal Therapeutics and Vaccines.

Fungal infections are considered a great threat to human life and are associated with high mortality and morbidity, especially in immunocompromised individuals. Fungal pathogens employ various defense mechanisms to evade the host immune system, which causes severe infections. The available repertoire of drugs for the treatment of fungal infections includes azoles, allylamines, polyenes, echinocandins, and antimetabolites. However, the development of multidrug and pandrug resistance to available antimycotic drugs increases the need to develop better treatment approaches. In this new era of -omics, bioinformatics has expanded options for treating fungal infections. This review emphasizes how bioinformatics complements the emerging strategies, including advancements in drug delivery systems, combination therapies, drug repurposing, epitope-based vaccine design, RNA-based therapeutics, and the role of gut-microbiome interactions to combat anti-fungal resistance. In particular, we focused on computational methods that can be useful to obtain potent hits, and that too in a short period.

Efavirenz: New Hope in Cancer Therapy.

Despite extensive research directed at preventive and treatment strategies, breast cancer remains the leading cause of cancer-related mortality among women. This necessitates the development of a new medication aimed at increasing patient survival and quality of life. A new drug's development from the ground up can cost billions of dollars and take up to ten or more years. Because much of the required safety and pharmacokinetic data are already available from earlier trials, repurposing medications usually results in lower costs and shorter turnaround times. Many antiretroviral medications target biological pathways and enzymes associated with cancer, which becomes an ideal option for repurposing as anticancer medications. Efavirenz is an antiretroviral medication that targets molecular pathways implicated in the growth of breast cancer, such as LINE-1 (long interspersed nuclear elements-1) suppression, hence lowering the proliferation of breast cancer cells and exhibiting anti-cancer properties. Additionally, it suppresses the fatty acid synthase gene and other important genes related to fat metabolism, impairing mitochondrial activity and making cancer cells deprived of energy. Efavirenz also inhibits cancer-initiating stem cells, promotes differentiation, and prevents recurrence. Additionally, efavirenz promotes oxidative damage by the formation of superoxide in cancer cells. In addition to its anti-cancer properties, efavirenz has the advantage of being a well-established and relatively inexpensive medication with a favorable safety profile. If proven effective, efavirenz could offer a cost-effective therapeutic option, which is an intriguing direction that warrants further investigation.

Challenges for developing bacterial CA inhibitors as novel antibiotics.

Acetazolamide, methazolamide, ethoxzolamide and dorzolamide, classical sulfonamide carbonic anhydrase (CA) inhibitors (CAIs) designed for targeting human enzymes, were also shown to effectively inhibit bacterial CAs and were proposed for repurposing as antibacterial agents against several infective agents. CAs belonging to the α-, ß- and/or γ-classes from pathogens such as Helicobacter pylori, Neisseria gonorrhoeae, vacomycin resistant enterococci (VRE), Vibrio cholerae, Mycobacterium tuberculosis, Pseudomonas aeruginosa and other bacteria were considered as drug targets for which several classes of potent inhibitors have been developed. Treatment of some of these pathogens with various classes of such CAIs led to an impairment of the bacterial growth, reduced virulence and for drug resistant bacteria, a resensitization to clinically used antibiotics. Here I will discuss the strategies and challenges for obtaining CAIs with enhanced selectivity for inhibiting bacterial versus human enzymes, which may constitute an important weapon for addressing the drug resistance to ß-lactams and other clinically used antibiotics.

Anticancer effect of the antipsychotic agent penfluridol on epithelial ovarian cancer.

OBJECTIVE: Chemoresistant-epithelial ovarian cancer (EOC) has a poor prognosis, prompting the search for new therapeutic drugs. The diphenylbutylpiperidine (DPBP) class of antipsychotic drugs used in schizophrenia has shown anticancer effects. This study aimed to investigate the preclinical efficacy of penfluridol, fluspirilene, and pimozide (DPBP) using in vitro and in vivo models of EOC. METHODS: Human EOC cell lines A2780, HeyA8, SKOV3ip1, A2780-CP20, HeyA8-MDR, and SKOV3-TR were treated with penfluridol, fluspirilene, and pimozide, and cell proliferation, apoptosis, and migration were assessed. The preclinical efficacy of DPBP was also investigated using in vivo mouse models, including cell lines and patient-derived xenografts (PDX) of EOC. RESULTS: DPBP drugs significantly decreased cell proliferation in chemosensitive (A2780, HeyA8, and SKOV3ip1) and chemoresistant (A2780-CP20, HeyA8-MDR, and SKOV3-TR) cell lines. Among these drugs, penfluridol exerted a relatively stronger cytotoxic effect on all cell lines. Penfluridol significantly increased apoptosis and inhibited migration of EOC cells. In the cell line xenograft mouse model with HeyA8, the penfluridol group showed significantly decreased tumor weight compared with the control group. In the paclitaxel-resistant model with HeyA8-MDR, the penfluridol group had significantly decreased tumor weight compared with the paclitaxel or control groups. Penfluridol exerted anticancer effects on the PDX model. CONCLUSION: Penfluridol exerted significant anticancer effects on EOC cells and xenograft models, including PDX. Thus, penfluridol therapy, as a drug repurposing strategy, might be a potential therapeutic for EOCs.

Alpha-1 antitrypsin inhibits Clostridium botulinum C2 toxin, Corynebacterium diphtheriae diphtheria toxin and B. anthracis fusion toxin.

The bacterium Clostridium botulinum, well-known for producing botulinum neurotoxins, which cause the severe paralytic illness known as botulism, produces C2 toxin, a binary AB-toxin with ADP-ribosyltranferase activity. C2 toxin possesses two separate protein components, an enzymatically active A-component C2I and the binding and translocation B-component C2II. After proteolytic activation of C2II to C2IIa, the heptameric structure binds C2I and is taken up via receptor-mediated endocytosis into the target cells. Due to acidification of endosomes, the C2IIa/C2I complex undergoes conformational changes and consequently C2IIa forms a pore into the endosomal membrane and C2I can translocate into the cytoplasm, where it ADP-ribosylates G-actin, a key component of the cytoskeleton. This modification disrupts the actin cytoskeleton, resulting in the collapse of cytoskeleton and ultimately cell death. Here, we show that the serine-protease inhibitor α1-antitrypsin (α1AT) which we identified previously from a hemofiltrate library screen for PT from Bordetella pertussis is a multitoxin inhibitor. α1AT inhibits intoxication of cells with C2 toxin via inhibition of binding to cells and inhibition of enzyme activity of C2I. Moreover, diphtheria toxin and an anthrax fusion toxin are inhibited by α1AT. Since α1AT is commercially available as a drug for treatment of the α1AT deficiency, it could be repurposed for treatment of toxin-mediated diseases.

In silico comparative analysis of cestode and human NPC1 provides insights for ezetimibe repurposing to visceral cestodiases treatment.

Visceral cestodiases, like cysticercoses and echinococcoses, are caused by cystic larvae from parasites of the Cestoda class and are endemic or hyperendemic in many areas of the world. Current therapeutic approaches for these diseases are complex and present limitations and risks. Therefore, new safer and more effective treatments are urgently needed. The Niemann-Pick C1 (NPC1) protein is a cholesterol transporter that, based on genomic data, would be the solely responsible for cholesterol uptake in cestodes. Considering that human NPC1L1 is a known target of ezetimibe, used in the treatment of hypercholesterolemia, it has the potential for repurposing for the treatment of visceral cestodiases. Here, phylogenetic, selective pressure and structural in silico analyses were carried out to assess NPC1 evolutive and structural conservation, especially between cestode and human orthologs. Two NPC1 orthologs were identified in cestode species (NPC1A and NPC1B), which likely underwent functional divergence, leading to the loss of cholesterol transport capacity in NPC1A. Comparative interaction analyses performed by molecular docking of ezetimibe with human NPC1L1 and cestode NPC1B pointed out to similarities that consolidate the idea of cestode NPC1B as a target for the repurposing of ezetimibe as a drug for the treatment of visceral cestodiases.

Drug Repurposing Approach to Identify Candidate Drug Molecules for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer, with a high mortality rate due to the limited therapeutic options. Systemic drug treatments improve the patient's life expectancy by only a few months. Furthermore, the development of novel small molecule chemotherapeutics is time-consuming and costly. Drug repurposing has been a successful strategy for identifying and utilizing new therapeutic options for diseases with limited treatment options. This study aims to identify candidate drug molecules for HCC treatment through repurposing existing compounds, leveraging the machine learning tool MDeePred. The Open Targets Platform, UniProt, ChEMBL, and Expasy databases were used to create a dataset for drug target interaction (DTI) predictions by MDeePred. Enrichment analyses of DTIs were conducted, leading to the selection of 6 out of 380 DTIs identified by MDeePred for further analyses. The physicochemical properties, lipophilicity, water solubility, drug-likeness, and medicinal chemistry properties of the candidate compounds and approved drugs for advanced stage HCC (lenvatinib, regorafenib, and sorafenib) were analyzed in detail. Drug candidates exhibited drug-like properties and demonstrated significant target docking properties. Our findings indicated the binding efficacy of the selected drug compounds to their designated targets associated with HCC. In conclusion, we identified small molecules that can be further exploited experimentally in HCC therapeutics. Our study also demonstrated the use of the MDeePred deep learning tool in in silico drug repurposing efforts for cancer therapeutics.

CEP-1347 Boosts Chk2-Mediated p53 Activation by Ionizing Radiation to Inhibit the Growth of Malignant Brain Tumor Cells.

Radiation therapy continues to be the cornerstone treatment for malignant brain tumors, the majority of which express wild-type p53. Therefore, the identification of drugs that promote the ionizing radiation (IR)-induced activation of p53 is expected to increase the efficacy of radiation therapy for these tumors. The growth inhibitory effects of CEP-1347, a known inhibitor of MDM4 expression, on malignant brain tumor cell lines expressing wild-type p53 were examined, alone or in combination with IR, by dye exclusion and/or colony formation assays. The effects of CEP-1347 on the p53 pathway, alone or in combination with IR, were examined by RT-PCR and Western blot analyses. The combination of CEP-1347 and IR activated p53 in malignant brain tumor cells and inhibited their growth more effectively than either alone. Mechanistically, CEP-1347 and IR each reduced MDM4 expression, while their combination did not result in further decreases. CEP-1347 promoted IR-induced Chk2 phosphorylation and increased p53 expression in concert with IR in a Chk2-dependent manner. The present results show, for the first time, that CEP-1347 is capable of promoting Chk2-mediated p53 activation by IR in addition to inhibiting the expression of MDM4 and, thus, CEP-1347 has potential as a radiosensitizer for malignant brain tumors expressing wild-type p53.