Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

Since their discovery over 5 decades ago, quinolone antibiotics have found enormous success as broad spectrum agents that exert their activity through dual inhibition of bacterial DNA gyrase and topoisomerase IV. Increasing rates of resistance, driven largely by target-based mutations in the GyrA/ParC quinolone resistance determining region, have eroded the utility and threaten the future use of this vital class of antibiotics. Herein we describe the discovery and optimization of a series of 4-(aminomethyl)quinolin-2(1H)-ones, exemplified by 34, that inhibit bacterial DNA gyrase and topoisomerase IV and display potent activity against ciprofloxacin-resistant Gram-negative pathogens. X-ray crystallography reveals that 34 occupies the classical quinolone binding site in the topoisomerase IV-DNA cleavage complex but does not form significant contacts with residues in the quinolone resistance determining region.

Mechanistic target of rapamycin (mTOR) is a fundamental regulator of cell growth, proliferation, and metabolism. mTOR is activated in renal cancer and accelerates tumor progression. Here, we report that the mTOR inhibitor, DEP domain-containing mTOR-interacting protein (DEPTOR), is strikingly suppressed in clear cell renal cell carcinoma (ccRCC) tumors and cell lines. We demonstrate that DEPTOR is repressed by both hypoxia-inducible factors, HIF-1 and HIF-2, which occurs through activation of the HIF-target gene and transcriptional repressor, BHLHe40/DEC1/Stra13. Restoration of DEPTOR- and CRISPR/Cas9-mediated knockout experiments demonstrate that DEPTOR is growth inhibitory in ccRCC. Furthermore, loss of DEPTOR confers resistance to second-generation mTOR kinase inhibitors through deregulated mTORC1 feedback to IRS-2/PI3K/Akt. This work reveals a hitherto unknown mechanism of resistance to mTOR kinase targeted therapy that is mediated by HIF-dependent reprograming of mTOR/DEPTOR networks and suggests that restoration of DEPTOR in ccRCC will confer sensitivity to mTOR kinase therapeutics.

Type II topoisomerases perform essential roles in DNA replication, chromosome segregation

Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. Certain strains of Klebsiella have become highly resistant to antibiotics. Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. In this paper, the structure of its DNA-cleavage complex is reported at 3.35 angstrom resolution. The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. This complex is compared with a similar complex from Streptococcus pneumoniae, which has recently been solved.

Klebsiella pneumoniae is a Gram-negative bacterium that is responsible for a range of common infections, including pulmonary pneumonia, bloodstream infections and meningitis. Certain strains of Klebsiella have become highly resistant to antibiotics. Despite the vast amount of research carried out on this class of bacteria, the molecular structure of its topoisomerase IV, a type II topoisomerase essential for catalysing chromosomal segregation, had remained unknown. In this paper, the structure of its DNA-cleavage complex is reported at 3.35 Å resolution. The complex is comprised of ParC breakage-reunion and ParE TOPRIM domains of K. pneumoniae topoisomerase IV with DNA stabilized by levofloxacin, a broad-spectrum fluoroquinolone antimicrobial agent. This complex is compared with a similar complex from Streptococcus pneumoniae , which has recently been solved.

Mechanistic target of rapamycin (mTOR) is an atypical Ser/Thr protein kinase that acts as a cellular hub integrating cell growth signals and nutrient availability to promote cell survival.1 mTOR is the catalytic component of 2 functionally distinct complexes (mTORC1 and mTORC2), and belongs to the family of phosphatidyl-inositol(3)-like kinases (PIKKs) together with ATM, ATR and DNA-PKcs. The latter 3 kinases have well established roles in the DNA damage response (DDR) – the signaling network activated by DNA damage to promote cell survival by enhancing DNA repair and delaying cell cycle progression in order to maximize repair. However, the participation of mTOR in these pathways has only recently been recognized, aided by the generation and availability of ATP-competitive mTOR kinase inhibitors. A better understanding of the regulation of the DDR is not only vital to understand aspects of tumor development, but is also important to improve DNA damage-based cancer therapies by targeting specific aspects, such as mTOR signaling. mTOR may act to promote cell survival either by regulating DNA repair and/or cell cycle arrest. Indeed, recent work has demonstrated a role for mTOR kinase in DNA repair through the regulation of FANCD2, a key protein involved in the repair of double-strand DNA breaks.2,3 Our recent work in Oncotarget4 also implicates mTOR in control of the cell cycle arrest aspect of the DDR. We found that mTOR is required for efficient establishment of S and G2/M cell cycle arrest after DNA damage, since ablation of mTOR resulted in a reduction in the percentage of cells in S and G2/M. Interestingly, we found that mTORC2 was required for the DNA damage-induced activation of Chk1, a key regulator of cell cycle arrest. We found that mTOR inhibition significantly inhibited all 3 DNA damage-induced phosphorylations of Chk1 (S296, S317 and S345) and reduced the production of Chk1 protein. Since mTOR inhibition consistently ablated DNA damage-induced Chk1 phosphorylation under all conditions tested, whereas inhibition of protein levels was cell type specific, this suggests that there may be distinct mechanisms by which mTOR controls Chk1 and that phosphorylation of Chk1 may be of primacy. Exactly how mTOR might regulate Chk1 activity is an open question. One likely candidate could be via ATR, since its ability to phosphorylate Chk1 at Ser317 and Ser345 leading to autophosphorylation at Ser296 is well established. Alternatively, the effects on Chk1 might involve one of the substrates of mTORC2. Our results support accumulating evidence from studies in yeast implicating TORC2 in survival under conditions of DNA damage and in genome stability.5 Since approval of the rapalog, Everolimus, in combination with hormone therapies for breast cancer, the potential of mTOR inhibitors to overcome resistance to current therapies has gained increasing interest. Indeed numerous publications have demonstrated that mTOR inhibition can also enhance radio- or chemotherapy-induced cell death in various cancer types. We also investigated the translational significance of our results and found that an mTOR kinase inhibitor enhanced DNA damage-induced cell death in HEK293 cells and a panel of breast cancer cell lines.4 While this suggests that mTOR kinase inhibitors may be useful therapeutically to augment DNA damage based therapies, it is likely that these effects will be tumor-type specific since similar augmentation was not found in renal and colon cancers.6,7 We have previously shown that an mTOR kinase inhibitor prevented p53-dependent cell death in renal carcinoma cells.6 It is known that mTORC1 contributes to DNA damage-induced p53 activation via multiple mechanisms, therefore it is likely that mTOR ablation leads to p53 suppression and inhibition of p53-dependent apoptosis in these renal cells. However, HEK293 cells have non-functional p53 and the panel of breast cancer cell lines that we tested displayed a variety of either wild-type, lost or mutated p53, but all still exhibited chemosensitivity following mTOR kinase inhibition. This suggests that these latter cells may rely on mTORC2-Chk1 signaling for survival. In conclusion, it is clear that mTOR regulates the DDR response by multiple mechanisms. Whether a given tumor type is made chemosensitive or chemoresistant by mTOR kinase inhibitors will depend on which aspect of mTOR signaling is altered during tumorigenesis. In this vein, it will be important to widen our understanding of mTOR targets following DNA damage in order to gain better insight into whether specific targeting of mTORC2 will be a useful adjuvant to DNA-damaging therapies.

mTOR signalling is commonly dysregulated in cancer. Concordantly, mTOR inhibitors have demonstrated efficacy in a subset of tumors and are in clinical trials as combination therapies. Although mTOR is associated with promoting cell survival after DNA damage, the exact mechanisms are not well understood. Moreover, since mTOR exists as two complexes, mTORC1 and mTORC2, the role of mTORC2 in cancer and in the DNA damage response is less well explored. Here, we report that mTOR protein levels and kinase activity are transiently increased by DNA damage in an ATM and ATR-dependent manner. We show that inactivation of mTOR with siRNA or pharmacological inhibition of mTORC1/2 kinase prevents etoposide-induced S and G2/M cell cycle arrest. Further results show that Chk1, a key regulator of the cell cycle arrest, is important for this since ablation of mTOR prevents DNA damage-induced Chk1 phosphorylation and decreases Chk1 protein production. Furthermore, mTORC2 was essential and mTORC1 dispensable, for this role. Importantly, we show that mTORC1/2 inhibition sensitizes breast cancer cells to chemotherapy. Taken together, these results suggest that breast cancer cells may rely on mTORC2-Chk1 pathway for survival and provide evidence that mTOR kinase inhibitors may overcome resistance to DNA-damage based therapies in breast cancer.

Bacterial drug resistance is a growing and now widely recognised threat and the limited number of new antibacterials developed in the recent years is a serious matter of concern. One of the approaches to combat this growing threat is to investigate the mechanisms of action of currently available antibacterials, as well as studying the way that bacteria are currently developing drug resistance and may potentially in the future develop drug resistance to known remedies. This knowledge should in turn be used in rational drug design and the general development of the appropriate frameworks for combating bacteria, while at the same time keeping the negative side effects of the drugs to the acceptable minimum. This is especially important when both bacteria and humans share similar drug targets, such as is the case for topoisomerases (in humans, the latter are also targeted by anti-cancer drugs). Our main protein targets of interest are type II topoisomerases which are involved in regulation of the DNA supercoiling in both bacteria and eukaryotes and also in decatenation of bacterial daughter chromosomes during cell division. Type II topoisomerases are performing their biological action by binding the double stranded DNA (called G-segment or Gate-DNA), temporarily cleaving it and passing another double stranded DNA (called T-segment) in the ATP-assisted process via the cleavage region thus changing the linking number in steps of 1 2. After that the G-segment is resealed and released. Several drugs were found to be able to disrupt this process ultimately resulting in the cell death (thus having anti-bacterial or anti-cancer action). Here we present our studies of the protein-DNA-drug interactions which are involved in the action of currently clinically used quinolone antibacterials as well as their newly developed alternatives (such as quinazolinediones) in relation to their mechanism of action and already established potential routes for developing drug resistance in bacteria. We present the results for different pathogens (including, but not limited to Streptococcus pneumoniae and Klebsiella pneumoniae) and also compare the configuration of the active site with the one from type II topoisomerases from a human organism.

Abstract Inactivating mutations in the von Hippel Lindau (VHL) tumour suppressor gene leads to both hereditary and sporadic clear cell renal cell carcinoma (ccRCC). Loss of VHL function results in stabilisation of hypoxia inducible factors 1 and 2 (HIF-1α and HIF-2α) with HIF-2α critical for renal tumour progression, angiogenesis and resistance to radiotherapy and chemotherapy. Previous work in VHL defective RCC cell lines that express HIF-2α has shown that loss of HIF-2α, either with siRNA or by reintroduction of VHL, increases phosphorylation and stabilisation of p53 which restored sensitivity to radiation and chemotherapy. Furthermore, as recent evidence suggests that mTORC2 is required for HIF-2α protein synthesis in VHL defective cells, we investigated the effects of pharmacological inhibition of HIF-2α with the mTORC1/2 kinase inhibitor pp242 alone or in combination with DNA damaging agents. Phosphorylated p53 and total p53 levels were significantly elevated in VHL competent RCC cells lacking HIF-2α as compared with VHL defective cells, confirming a role for HIF-2α mediated inhibition of p53. We analysed the effects of the mTORC1/2 kinase inhibitor pp242 on HIF-2α protein levels by Western blot. pp242 inhibited phosphorylation of both mTOR and its substrate p70S6K and significantly reduced protein levels of HIF-2α as compared with the mTORC1 inhibitor rapamycin, which had no effect on HIF-2α. Since the DNA damaging agent camptothecin (CPT) is a potent inhibitor of HIF-1α we also determined its effects on HIF-2α expression. CPT reduced HIF-2α protein levels in VHL defective RCC cells and increased p53 stabilisation, which was significantly augmented in VHL competent cells following complete loss of HIF-2α. Consistent with its inhibition of the p53 pathway, HIF-2α prevented both spontaneous and CPT induced apoptosis of RCC cells as demonstrated by FACS analysis of cells in sub G1 and Western blot analysis of cleaved PARP. Taken together these results confirm that HIF-2α negatively regulates p53, confers resistance to DNA damage induced apoptosis in VHL defective RCC cell lines and that further studies investigating the pharmacological inhibition of HIF-2α mediated by mTORC1/2 kinase inhibitors alone or in combination with DNA damaging agents for the treatment of ccRCC are warranted. Citation Format: Jogitha Selvarajah, Abdeladim Moumen, Veronica A. Carroll. Targeting the mTORC2/HIF-2alpha/p53 pathway in clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5088. doi:10.1158/1538-7445.AM2013-5088

The DDR (DNA damage response) is a signalling transduction cascade utilizing many forms of post-translation modification of proteins, including phosphorylation and ubiquitination. The well-known function of ubiquitination is to target proteins for proteasomal degradation; however, it is also involved in the regulation of protein function. The present review describes how ubiquitination regulates the function of certain proteins involved in DDR, in particular FANCD2 (Fanconi's anaemia complementation group D2) and PCNA (proliferating-cell nuclear antigen). Also, the proteomic methods currently used to identify new ubiquitinated proteins in response to DNA damage, including the advantages of using the UBD (ubiquitin-binding domain) beads to purify the ubiquitinated proteins, are considered.