Engineered Bacillus subtilis for the de novo production of 2'-fucosyllactose.

BACKGROUND: The most abundant human milk oligosaccharide in breast milk, 2'-fucosyllactose (2'-FL), has been approved as an additive to infant formula due to its multifarious nutraceutical and pharmaceutical functions in promoting neonate health. However, the low efficiency of de novo synthesis limits the cost-efficient bioproduction of 2'-FL. RESULTS: This study achieved 2'-FL de novo synthesis in a generally recognized as safe (GRAS) strain Bacillus subtilis. First, a de novo biosynthetic pathway for 2'-FL was introduced by expressing the manB, manC, gmd, wcaG, and futC genes from Escherichia coli and Helicobacter pylori in B. subtilis, resulting in 2'-FL production of 1.12 g/L. Subsequently, a 2'-FL titer of 2.57 g/L was obtained by reducing the competitive lactose consumption, increasing the regeneration of the cofactor guanosine-5'-triphosphate (GTP), and enhancing the supply of the precursor mannose-6-phosphate (M6P). By replacing the native promoter of endogenous manA gene (encoding M6P isomerase) with a constitutive promoter P7, the 2'-FL titer in shake flask reached 18.27 g/L. The finally engineered strain BS21 could produce 88.3 g/L 2'-FL with a yield of 0.61 g/g lactose in a 3-L bioreactor, without the addition of antibiotics and chemical inducers. CONCLUSIONS: The efficient de novo synthesis of 2'-FL can be achieved by the engineered B. subtilis, paving the way for the large-scale bioproduction of 2'-FL titer in the future.

In silico screening identifies a novel small molecule inhibitor that counteracts PARP inhibitor resistance in ovarian cancer.

Poly ADP-ribose polymerase (PARP) inhibitors are promising targeted therapy for epithelial ovarian cancer (EOC) with BRCA mutations or defective homologous recombination (HR) repair. However, reversion of BRCA mutation and restoration of HR repair in EOC lead to PARP inhibitor resistance and reduced clinical efficacy of PARP inhibitors. We have previously shown that triapine, a small molecule inhibitor of ribonucleotide reductase (RNR), impaired HR repair and sensitized HR repair-proficient EOC to PARP inhibitors. In this study, we performed in silico screening of small molecule libraries to identify novel compounds that bind to the triapine-binding pocket on the R2 subunit of RNR and inhibit RNR in EOC cells. Following experimental validation of selected top-ranking in silico hits for inhibition of dNTP and DNA synthesis, we identified, DB4, a putative RNR pocket-binding inhibitor markedly abrogated HR repair and sensitized BRCA-wild-type EOC cells to the PARP inhibitor olaparib. Furthermore, we demonstrated that the combination of DB4 and olaparib deterred the progression of BRCA-wild type EOC xenografts and significantly prolonged the survival time of tumor-bearing mice. Herein we report the discovery of a putative small molecule inhibitor of RNR and HR repair for combination with PARP inhibitors to treat PARP inhibitor-resistant and HR repair-proficient EOC.

Pyruvate-responsive genetic circuits for dynamic control of central metabolism.

Dynamic regulation is a promising strategy for fine-tuning metabolic fluxes in microbial cell factories. However, few of these synthetic regulatory systems have been developed for central carbon metabolites. Here we created a set of programmable and bifunctional pyruvate-responsive genetic circuits for dynamic dual control (activation and inhibition) of central metabolism in Bacillus subtilis. We used these genetic circuits to design a feedback loop control system that relies on the intracellular concentration of pyruvate to fine-tune the target metabolic modules, leading to the glucaric acid titer increasing from 207 to 527 mg l-1. The designed logic gate-based circuits were enabled by the characterization of a new antisense transcription mechanism in B. subtilis. In addition, a further increase to 802 mg l-1 was achieved by blocking the formation of by-products. Here, the constructed pyruvate-responsive genetic circuits are presented as effective tools for the dynamic control of central metabolism of microbial cell factories.

Engineering Yarrowia lipolytica as a Chassis for De Novo Synthesis of Five Aromatic-Derived Natural Products and Chemicals.

Yarrowia lipolytica is a novel microbial chassis to upgrade renewable low-cost carbon feedstocks to high-value commodity chemicals and natural products. In this work, we systematically characterized and removed the rate-limiting steps of the shikimate pathway and achieved de novo synthesis of five aromatic chemicals in Y. lipolytica. We determined that eliminating amino acids formation and engineering feedback-insensitive DAHP synthases are critical steps to mitigate precursor competition and relieve the feedback regulation of the shikimate pathway. Further overexpression of heterologous phosphoketolase and deletion of pyruvate kinase provided a sustained metabolic driving force that channels E4P (erythrose 4-phosphate) and PEP (phosphoenolpyruvate) precursors through the shikimate pathway. Precursor competing pathways and byproduct formation pathways were also blocked by inactivating chromosomal genes. To demonstrate the utility of our engineered chassis strain, three natural products, 2-phenylethanol (2-PE), p-coumaric acid, and violacein, which were derived from phenylalanine, tyrosine, and tryptophan, respectively, were chosen to test the chassis performance. We obtained 2426.22 ± 48.33 mg/L of 2-PE, 593.53 ± 28.75 mg/L of p-coumaric acid, 12.67 ± 2.23 mg/L of resveratrol, 366.30 ± 28.99 mg/L of violacein, and 55.12 ± 2.81 mg/L of deoxyviolacein from glucose in a shake flask. The 2-PE production represents a 286-fold increase over the initial strain (8.48 ± 0.50 mg/L). Specifically, we obtained the highest 2-PE, violacein, and deoxyviolacein titer ever reported from the de novo shikimate pathway in yeast. These results set up a new stage of engineering Y. lipolytica as a sustainable biorefinery chassis strain for de novo synthesis of aromatic compounds with economic values.

Refactoring Ehrlich Pathway for High-Yield 2-Phenylethanol Production in Yarrowia lipolytica.

Efficient microbial synthesis of chemicals requires the coordinated supply of precursors and cofactors to maintain cell growth and product formation. Substrates with different entry points into the metabolic network have different energetic and redox statuses. Generally, substrate cofeeding could bypass the lengthy and highly regulated native metabolism and facilitates high carbon conversion rate. Aiming to efficiently synthesize the high-value rose-smell 2-phenylethanol (2-PE) in Y. lipolytica, we analyzed the stoichiometric constraints of the Ehrlich pathway and identified that the selectivity of the Ehrlich pathway and the availability of 2-oxoglutarate are the rate-limiting factors. Stepwise refactoring of the Ehrlich pathway led us to identify the optimal catalytic modules consisting of l-phenylalanine permease, ketoacid aminotransferase, phenylpyruvate decarboxylase, phenylacetaldehyde reductase, and alcohol dehydrogenase. On the other hand, mitochondrial compartmentalization of 2-oxoglutarate inherently creates a bottleneck for efficient assimilation of l-phenylalanine, which limits 2-PE production. To improve 2-oxoglutarate (aKG) trafficking across the mitochondria membrane, we constructed a cytosolic aKG source pathway by coupling a bacterial aconitase with a native isocitrate dehydrogenase (ylIDP2). Additionally, we also engineered dicarboxylic acid transporters to further improve the 2-oxoglutarate availability. Furthermore, by blocking the precursor-competing pathways and mitigating fatty acid synthesis, the engineered strain produced 2669.54 mg/L of 2-PE in shake flasks, a 4.16-fold increase over the starting strain. The carbon conversion yield reaches 0.702 g/g from l-phenylalanine, 95.0% of the theoretical maximal. The reported work expands our ability to harness the Ehrlich pathway for production of high-value aromatics in oleaginous yeast species.

Combination of triapine, olaparib, and cediranib suppresses progression of BRCA-wild type and PARP inhibitor-resistant epithelial ovarian cancer.

PARP inhibitors target BRCA mutations and defective homologous recombination repair (HRR) for the treatment of epithelial ovarian cancer (EOC). However, the treatment of HRR-proficient EOC with PARP inhibitors remains challenging. The objective of this study was to determine whether the combination of triapine (ribonucleotide reductase inhibitor), cediranib (vascular endothelial growth factor receptor tyrosine kinase inhibitor), and the PARP inhibitor olaparib synergized against BRCA wild-type and HRR-proficient EOC in xenograft mouse models. In addition, the mechanisms by which cediranib augmented the efficacy of triapine and olaparib were investigated. BRCA-wild type and PARP inhibitor-resistant EOC cell lines were implanted subcutaneously (s.c.) into nude mice or injected intraperitoneally (i.p.) into SCID-Beige mice. Mice were then treated i.p. with olaparib, cediranib, triapine, various double and triple combinations. The volume of s.c tumor in nude mice and the abdominal circumference of SCID-Beige mice were measured to evaluate the effectiveness of the treatment to delay tumor growth and prolong the survival time of mice. In both xenograft mouse models, the combination of triapine, olaparib and cediranib resulted in marked suppression of BRCA-wild type EOC growth and significant prolongation of the survival time of mice, with efficacy greater than any double combinations and single drugs. Furthermore, we identified that cediranib abrogated pro-survival and anti-apoptotic AKT signaling, thereby enhancing the efficacy of triapine and olaparib against BRCA-wild type EOC cells. Taken together, our results demonstrate a proof-of-principle approach and the combination regiment holds promise in treating BRCA-wild type and PARP inhibitor-resistant EOC.

Targeting Cyclin-Dependent Kinases for Treatment of Gynecologic Cancers.

Ovarian, uterine/endometrial, and cervical cancers are major gynecologic malignancies estimated to cause nearly 30,000 deaths in 2018 in US. Defective cell cycle regulation is the hallmark of cancers underpinning the development and progression of the disease. Normal cell cycle is driven by the coordinated and sequential rise and fall of cyclin-dependent kinases (CDK) activity. The transition of cell cycle phases is governed by the respective checkpoints that prevent the entry into the next phase until cellular or genetic defects are repaired. Checkpoint activation is achieved by p53- and ATM/ATR-mediated inactivation of CDKs in response to DNA damage. Therefore, an aberrant increase in CDK activity and/or defects in checkpoint activation lead to unrestricted cell cycle phase transition and uncontrolled proliferation that give rise to cancers and perpetuate malignant progression. Given that CDK activity is also required for homologous recombination (HR) repair, pharmacological inhibition of CDKs can be exploited as a synthetic lethal approach to augment the therapeutic efficacy of PARP inhibitors and other DNA damaging modalities for the treatment of gynecologic cancers. Here, we overview the basic of cell cycle and discuss the mechanistic studies that establish the intimate link between CDKs and HR repair. In addition, we present the perspective of preclinical and clinical development in small molecule inhibitors of CDKs and CDK-associated protein targets, as well as their potential use in combination with hormonal therapy, PARP inhibitors, chemotherapy, and radiation to improve treatment outcomes.

Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive ovarian carcinoma cell lines.

Although epithelial ovarian cancers (EOCs) are initially treated with platinum-based chemotherapy, EOCs vary in platinum responsiveness. Cataloging antineoplastic agents according to their effectiveness against platinum-resistant and platinum-sensitive EOC cell lines is valuable for development of therapeutic strategies to avoid platinum inefficacy and to exploit platinum sensitivity. TOV-21G devoid of FANCF expression, OV-90 and SKOV-3 were employed as examples of platinum-sensitive, platinum-intermediate and platinum-resistant cell lines, respectively. Antineoplastic agents examined included mitomycin C, doxorubicin, etoposide, gemcitabine, chlorambucil, paclitaxel, triapine and X-rays. Their effectiveness against cell lines was analyzed by clonogenic assays. Cytotoxic profiles of mitomycin C and carboplatin were similar, with mitomycin C exhibiting greater potency and selectivity against TOV-21G than carboplatin. Cytotoxic profiles of doxorubicin, etoposide and X-rays overlapped with that of carboplatin, while OV-90 overexpressing Rad51 was more resistant to chlorambucil than SKOV-3. The efficacy of paclitaxel and triapine was independent of platinum sensitivity or resistance. Consistent with these cytotoxic profiles, cisplatin/mitomycin C, triapine, and paclitaxel differed in the capacity to induce phosphorylation of H2AX, and produced unique inhibitory patterns of DNA/RNA syntheses in HL-60 human leukemia cells. Paclitaxel and triapine in combination produced additive antitumor effects in M109 murine lung carcinoma. In conclusion, mitomycin C is potentially more effective against Fanconi anemia pathway-deficient EOCs than carboplatin. Doxorubicin and etoposide, because of their overlapping cytotoxic properties with carboplatin, are unlikely to be efficacious against platinum-refractory EOCs. Paclitaxel and triapine are effective regardless of platinum sensitivity status, and promising in combination for both platinum-sensitive and platinum-refractory EOCs.

Triapine potentiates platinum-based combination therapy by disruption of homologous recombination repair.

BACKGROUND: Platinum resistance may be attributable to inherent or acquired proficiency in homologous recombination repair (HRR) in epithelial ovarian cancer (EOC). The objective of this study was to evaluate the efficacy of the small molecule inhibitor triapine to disrupt HRR and sensitise BRCA wild-type EOC cells to platinum-based combination therapy in vitro and in vivo. METHODS: The sensitivity of BRCA wild-type cancer cells to olaparib, cisplatin, carboplatin, doxorubicin, or etoposide in combination with triapine was evaluated by clonogenic survival assays. The effects of triapine on HRR activity in cells were measured with a DR-GFP reporter assay. The ability of triapine to enhance the effects of the carboplatin-doxil combination on EOC tumour growth delay was determined using a xenograft tumour mouse model. RESULTS: Platinum resistance is associated with wild-type BRCA status. Triapine inhibits HRR activity and enhances the sensitivity of BRCA wild-type cancer cells to cisplatin, olaparib, and doxorubicin. However, sequential combination of triapine and cisplatin is necessary to achieve synergism. Moreover, triapine potentiates platinum-based combination therapy against BRCA wild-type EOC cells and produces significant delay of EOC tumour growth. CONCLUSIONS: Triapine promises to augment the clinical efficacy of platinum-based combination regimens for treatment of platinum-resistant EOC with wild-type BRCA and proficient HRR activity.

Antitumor sulfonylhydrazines: design, structure-activity relationships, resistance mechanisms, and strategies for improving therapeutic utility.

1,2-Bis(sulfonyl)-1-alkylhydrazines (BSHs) were conceived as more specific DNA guanine O-6 methylating and chloroethylating agents lacking many of the undesirable toxicophores contained in antitumor nitrosoureas. O(6)-Alkylguanine-DNA alkyltransferase (MGMT) is the sole repair protein for O(6)-alkylguanine lesions in DNA and has been reported to be absent in 5-20% of most tumor types. Many BSHs exhibit highly selective cytotoxicity toward cells deficient in MGMT activity. The development of clinically useful MGMT assays should permit the identification of tumors with this vulnerability and allow for the preselection of patient subpopulations with a high probability of responding. The BSH system is highly versatile, permitting the synthesis of many prodrug types with the ability to incorporate an additional level of tumor-targeting due to preferential activation by tumor cells. Furthermore, it may be possible to expand the spectrum of activity of these agents to include tumors with MGMT activity by combining them with tumor-targeted MGMT inhibitors.

Distinct mechanisms of cell-kill by triapine and its terminally dimethylated derivative Dp44mT due to a loss or gain of activity of their copper(II) complexes.

Triapine, currently being evaluated as an antitumor agent in phase II clinical trials, and its terminally dimethylated derivative Dp44mT share the α-pyridyl thiosemicarbazone backbone that functions as ligands for transition metal ions. Yet, Dp44mT is approximately 100-fold more potent than triapine in cytotoxicity assays. The aims of this study were to elucidate the mechanisms underlying their potency disparity and to determine their kinetics of cell-kill in culture to aid in the formulation of their clinical dosing schedules. The addition of Cu(2+) inactivated triapine in a 1:1 stoichiometric fashion, while it potentiated the cytotoxicity of Dp44mT. Clonogenic assays after finite-time drug-exposure revealed that triapine produced cell-kill in two phases, one completed within 20 min that caused limited cell-kill, and the other occurring after 16 h of exposure that produced extensive cell-kill. The ribonucleotide reductase inhibitor triapine at 0.4 µM caused immediate complete arrest of DNA synthesis, whereas Dp44mT at this concentration did not appreciably inhibit DNA synthesis. The inhibition of DNA synthesis by triapine was reversible upon its removal from the medium. Cell death after 16 h exposure to triapine paralleled the appearance of phospho-(γ)H2AX, a marker of DNA double-strand breaks induced by collapse of DNA replication forks after prolonged replication arrest. In contrast to triapine, Dp44mT produced robust cell-kill within 1h in a concentration-dependent manner. The short-term action of both agents was prevented by thiols, indicative of the involvement of reactive oxygen species. The time dependency in the production of cell-kill by triapine should be considered in treatment regimens.

Anti-tumor effects of adenovirus containing human growth hormone sequences in a mouse model of human ovarian cancer.

Women with ovarian cancer have a low survival rate and develop resistance to chemotherapy, so new approaches to treatment are needed. We unexpectedly found administration of a replication-deficient adenovirus containing human growth hormone sequences (AdXGH) was beneficial in a mouse model of human ovarian cancer. Intraperitoneal injections of AdXGH prolonged median survival from a mean of 31 ± 1.2 to 40 ± 1.4 days in immunodeficient SCID mice given SKOV3.ip1 human ovarian cancer cells in the peritoneal cavity. Adenovirus containing human prolactin or del32-71growth hormone sequences had no effect. Repeated injection of growth hormone or implantation of tablets with sustained growth hormone release did not increase survival. Control mice had overlapping tumors throughout the peritoneal cavity and liver and frequent lung metastases 24 days after tumor cell injection. Mice that received two injections of AdXGH had no lung metastases. Mice that received four injections had no lung or liver metastases and peritoneal fibrosis. They did not survive longer than mice that received two injections, but they had enlarged livers with hepatocellular changes, indicating that a limitation of increasing the dose is liver toxicity.

Quantitative relationship between guanine O(6)-alkyl lesions produced by Onrigin™ and tumor resistance by O(6)-alkylguanine-DNA alkyltransferase.

O(6)-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O(6)-chloroethyl (Onrigin™ and carmustine) and O(6)-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O(6)-methylguanines/cell and ∼300 O(6)-chloroethylguanines/cell, respectively. AGT repaired O(6)-methylguanines until the AGT pool was exhausted, while its repair of O(6)-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O(6)-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O(6)-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.

Pharmacokinetics analysis of sustained release hGH biodegradable implantable tablets using a mouse model of human ovarian cancer.

This paper presents the pharmacokinetic of human growth hormone (hGH) implantable tablets tested on a human ovarian cancer mouse model. In order to obtain a sustained release device which permits to administer a high dose of the hormone that keeps its integrity and stability, three different formulations of hGH-poly (d,l-lactic-co-glycolic acid) (PLGA) were elaborated by direct compression method varying hormone load, PLGA content and compactation time. In vitro studies showed that drug release was mainly controlled by hormone load. Pharmacokinetic studies were conducted by using immunodeficient female mice. Four days before the insertion of hGH implantable tablets in the peritoneal cavity, every mouse received 5x10(6) human ovarian cancer cells (SKOV3.ip1). Hormone serum levels were monitored through bleeding from eye orbital vessels. The population pharmacokinetic model used was based on the in series tank model and model parameters were estimated using the maximum likelihood method. The null hypothesis test about differences between formulations leads us to the conclusion that the three formulations showed the same kinetic behavior except for the hGH load. The hormone release was extended all over 2 weeks but no increase or decrease in survival time was observed. These results suggest that hGH serum levels do not facilitate tumoral cells proliferation, an expected effect of hGH and this could explain why survival times of mice treated with implantable tablets are not shorter than those treated with the control ones.

Disruption of cAMP and prostaglandin E2 transport by multidrug resistance protein 4 deficiency alters cAMP-mediated signaling and nociceptive response.

Multidrug resistance protein 4 (MRP4; ABCC4) is a member of the MRP/ATP-binding cassette family serving as a transmembrane transporter involved in energy-dependent efflux of anticancer/antiviral nucleotide agents and of physiological substrates, including cyclic nucleotides and prostaglandins (PGs). Phenotypic consequences of mrp4 deficiency were investigated using mrp4-knockout mice and derived immortalized mouse embryonic fibroblast (MEF) cells. Mrp4 deficiency caused decreased extracellular and increased intracellular levels of cAMP in MEF cells under normal and forskolin-stimulated conditions. Mrp4 deficiency and RNA interference-mediated mrp4 knockdown led to a pronounced reduction in extracellular PGE(2) but with no accumulation of intracellular PGE(2) in MEF cells. This result was consistent with attenuated cAMP-dependent protein kinase activity and reduced cyclooxygenase-2 (Cox-2) expression in mrp4-deficient MEF cells, suggesting that PG synthesis is restrained along with a lack of PG transport caused by mrp4 deficiency. Mice lacking mrp4 exhibited no outward phenotypes but had a decrease in plasma PGE metabolites and an increase in inflammatory pain threshold compared with wild-type mice. Collectively, these findings imply that mrp4 mediates the efflux of PGE(2) and concomitantly modulates cAMP mediated signaling for balanced PG synthesis in MEF cells. Abrogation of mrp4 affects the regulation of peripheral PG levels and consequently alters inflammatory nociceptive responses in vivo.