Indisulam is an aryl sulfonamide drug with selective anticancer activity. Its mechanism of action and the basis for its selectivity have so far been unknown. Here we show that indisulam promotes the ...recruitment of RBM39 (RNA binding motif protein 39) to the CUL4-DCAF15 E3 ubiquitin ligase, leading to RBM39 polyubiquitination and proteasomal degradation. Mutations in RBM39 that prevent its recruitment to CUL4-DCAF15 increase RBM39 stability and confer resistance to indisulam's cytotoxicity. RBM39 associates with precursor messenger RNA (pre-mRNA) splicing factors, and inactivation of RBM39 by indisulam causes aberrant pre-mRNA splicing. Many cancer cell lines derived from hematopoietic and lymphoid lineages are sensitive to indisulam, and their sensitivity correlates with DCAF15 expression levels. Two other clinically tested sulfonamides, tasisulam and chloroquinoxaline sulfonamide, share the same mechanism of action as indisulam. We propose that DCAF15 expression may be a useful biomarker to guide clinical trials of this class of drugs, which we refer to as SPLAMs (splicing inhibitor sulfonamides).
Overexpression of ATP-binding cassette (ABC) transporters is often linked to multidrug resistance (MDR) in cancer chemotherapies. P-glycoprotein (P-gp) is one of the best studied drug transporters ...associated with MDR. There are currently no approved drugs available for clinical use in cancer chemotherapies to reverse MDR by inhibiting P-glycoprotein. Using computational studies, we previously identified several compounds that inhibit P-gp by targeting its nucleotide binding domain and avoiding its drug binding domains. Several of these compounds showed successful MDR reversal when tested on a drug resistant prostate cancer cell line. Using conventional two-dimensional cell culture of MDR ovarian and prostate cancer cells and three dimensional prostate cancer microtumor spheroids, we demonstrated here that co-administration with chemotherapeutics significantly decreased cell viability and survival as well as cell motility. The P-gp inhibitors were not observed to be toxic on their own. The inhibitors increased cellular retention of chemotherapeutics and reporter compounds known to be transport substrates of P-gp. We also showed that these compounds are not transport substrates of P-gp and that two of the three inhibit P-gp, but not the closely related ABC transporter, ABCG2/BCRP. The results presented suggest that these P-gp inhibitors may be promising leads for future drug development.
Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is ...triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.
Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic ...reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.
The pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By ...screening a diverse synthetic chemical library, we have discovered two new classes of small molecules that disrupt Wnt pathway responses; whereas one class inhibits the activity of Porcupine, a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, which are suppressors of Wnt/beta-catenin pathway activity. With these small molecules, we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/beta-catenin pathway response in vivo, and we establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.
Correlation of mean %PPB in mouse plasma of 27 compounds determined by ultrafiltration versus rapid equilibrium dialysis. %PPB was determined by mass balance as described in Eq. (2) using ...ultrafiltration or as described in Eq. (1) using RED for twenty seven known and investigational compounds in mouse plasma. The values were plotted and a linear regression line plotted with the y intercept set to 0. Compounds deviating most significantly from the regression line are indicated by a lighter degree of shading and are discussed in the text. Display omitted
► The calculation of non-specific binding in PBS during ultrafiltration is challenged. ► Mass balance analysis shows recovery is significantly different in plasma versus PBS. ► Total compound recovery in plasma is a more valid measure of method utility. ► This approach shows good concordance with other plasma protein binding methods. ► The speed and ease of ultrafiltration avoids pitfalls seen with other methods.
The aim of this study is to further validate the use of ultrafiltration (UF) as a method for determining plasma protein binding (PPB) by demonstrating that non-specific binding (NSB) is not a limitation, even for highly lipophilic compounds, because NSB sites on the apparatus are passivated in the presence of plasma. Mass balance theory was used to calculate recovery of 20 commercial and seven investigational compounds during ultrafiltration in the presence and absence of plasma. PPB was also measured using this mass balance approach for comparison to PPB determined by rapid equilibrium dialysis (RED) and as found in the literature. Compound recovery during UF was dramatically different in the presence and absence of plasma for compounds with high NSB in PBS only. A comparison of PPB calculated by ultrafiltration with literature values or calculated by RED gave concordant results. Discrepancies could be explained by changes in pH, insufficient time to equilibrium, or compound instability during RED, problems which were circumvented by ultrafiltration. Therefore, NSB, as measured by the traditional incubation of compound in PBS, need not be an issue when choosing UF as a PPB assay method. It is more appropriate to calculate compound recovery from the device in plasma as measured by mass balance to determine the suitability of the method for an individual compound. The speed with which UF can be conducted additionally avoids changes in pH or compound loss that can occur with other methods. The mass balance approach to UF is thus a preferred method for rapid determination of PPB.
Summary Background DSM265 is a novel antimalarial that inhibits plasmodial dihydroorotate dehydrogenase, an enzyme essential for pyrimidine biosynthesis. We investigated the safety, tolerability, and ...pharmacokinetics of DSM265, and tested its antimalarial activity. Methods Healthy participants aged 18–55 years were enrolled in a two-part study: part 1, a single ascending dose (25–1200 mg), double-blind, randomised, placebo-controlled study, and part 2, an open-label, randomised, active-comparator controlled study, in which participants were inoculated with Plasmodium falciparum induced blood-stage malaria (IBSM) and treated with DSM265 (150 mg) or mefloquine (10 mg/kg). Primary endpoints were DSM265 safety, tolerability, and pharmacokinetics. Randomisation lists were created using a validated, automated system. Both parts were registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12613000522718 (part 1) and number ACTRN12613000527763 (part 2). Findings In part 1, 73 participants were enrolled between April 12, 2013, and July 14, 2015 (DSM265, n=55; placebo, n=18). In part 2, nine participants were enrolled between Sept 30 and Nov 25, 2013 (150 mg DSM265, n=7; 10 mg/kg mefloquine, n=2). In part 1, 117 adverse events were reported; no drug-related serious or severe events were reported. The most common drug-related adverse event was headache. The mean DSM265 peak plasma concentration (Cmax ) ranged between 1310 ng/mL and 34 800 ng/mL and was reached in a median time (tmax ) between 1·5 h and 4 h, with a mean elimination half-life between 86 h and 118 h. In part 2, the log10 parasite reduction ratio at 48 h in the DSM265 (150 mg) group was 1·55 (95% CI 1·42–1·67) and in the mefloquine (10 mg/kg) group was 2·34 (2·17–2·52), corresponding to a parasite clearance half-life of 9·4 h (8·7–10·2) and 6·2 h (5·7–6·7), respectively. The median minimum inhibitory concentration of DSM265 in blood was estimated as 1040 ng/mL (range 552–1500), resulting in a predicted single efficacious dose of 340 mg. Parasite clearance was significantly faster in participants who received mefloquine than in participants who received DSM265 (p<0·0001). Interpretation The good safety profile, long elimination half-life, and antimalarial effect of DSM265 supports its development as a partner drug in a single-dose antimalarial combination treatment. Funding Wellcome Trust, UK Department for International Development, Global Health Innovative Technology Fund, Bill & Melinda Gates Foundation.
The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory ...evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.
Therapeutic drugs that block DNA repair, including poly(ADP-ribose) polymerase (PARP) inhibitors, fail due to lack of tumor-selectivity. When PARP inhibitors and β-lapachone are combined, synergistic ...antitumor activity results from sustained NAD(P)H levels that refuel NQO1-dependent futile redox drug recycling. Significant oxygen-consumption-rate/reactive oxygen species cause dramatic DNA lesion increases that are not repaired due to PARP inhibition. In NQO1+ cancers, such as non-small-cell lung, pancreatic, and breast cancers, cell death mechanism switches from PARP1 hyperactivation-mediated programmed necrosis with β-lapachone monotherapy to synergistic tumor-selective, caspase-dependent apoptosis with PARP inhibitors and β-lapachone. Synergistic antitumor efficacy and prolonged survival were noted in human orthotopic pancreatic and non-small-cell lung xenograft models, expanding use and efficacy of PARP inhibitors for human cancer therapy.
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•Solid cancers overexpress NQO1, with low catalase levels, reverse of normal tissue•β-Lapachone kills independent of oncogenic driver or passenger mutations•Synergy with PARP inhibitors and β-lapachone is NQO1 dependent•PARP inhibitors + β-lap induce DNA lesions, block repair, and cause apoptosis
Huang et al. show that combination treatment with NQO1 bioactivatible β-lapachone and a PARP inhibitor causes unrepaired DNA damage and induces apoptosis, and that the combination treatment has a synergistic therapeutic effect in orthotopic pancreatic and non-small-cell lung cancer models.
Candida auris is an emerging fungal pathogen that exhibits resistance to multiple drugs, including the most commonly prescribed antifungal, fluconazole. Here, we use a combinatorial screening ...approach to identify a bis-benzodioxolylindolinone (azoffluxin) that synergizes with fluconazole against C. auris. Azoffluxin enhances fluconazole activity through the inhibition of efflux pump Cdr1, thus increasing intracellular fluconazole levels. This activity is conserved across most C. auris clades, with the exception of clade III. Azoffluxin also inhibits efflux in highly azole-resistant strains of Candida albicans, another human fungal pathogen, increasing their susceptibility to fluconazole. Furthermore, azoffluxin enhances fluconazole activity in mice infected with C. auris, reducing fungal burden. Our findings suggest that pharmacologically targeting Cdr1 in combination with azoles may be an effective strategy to control infection caused by azole-resistant isolates of C. auris.
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