Exploring the Mutated Kinases for Chemoenzymatic Synthesis of N4-Modified Cytidine Monophosphates.

Nucleosides, nucleotides, and their analogues are an important class of molecules that are used as substrates in research of enzymes and nucleic acid, or as antiviral and antineoplastic agents. Nucleoside phosphorylation is usually achieved with chemical methods; however, enzymatic phosphorylation is a viable alternative. Here, we present a chemoenzymatic synthesis of modified cytidine monophosphates, where a chemical synthesis of novel N4-modified cytidines is followed by an enzymatic phosphorylation of the nucleosides by nucleoside kinases. To enlarge the substrate scope, multiple mutant variants of Drosophila melanogaster deoxynucleoside kinase (DmdNK) (EC:2.7.1.145) and Bacillus subtilis deoxycytidine kinase (BsdCK) (EC:2.7.1.74) have been created and tested. It has been determined that certain point mutations in the active sites of the kinases alter their substrate specificities noticeably and allow phosphorylation of compounds that had been otherwise not phosphorylated by the wild-type DmdNK or BsdCK.

Biosynthesis of UDP-α-N-Acetyl-d-mannosaminuronic Acid and CMP-ß-N-Acetyl-d-neuraminic Acid for the Capsular Polysaccharides of Campylobacter jejuni.

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in North America and Europe. The exterior surface of the bacterial cell wall is attached to a polymeric coat of sugar molecules known as the capsular polysaccharide (CPS) that helps protect the organism from the host immune response. The CPS is composed of a repeating sequence of common and unusual sugar residues. In the HS:11 serotype of C. jejuni, we identified two enzymes in the gene cluster for CPS formation that are utilized for the biosynthesis of UDP-α-N-acetyl-d-mannosaminuronic acid (UDP-ManNAcA). In the first step, UDP-α-N-acetyl-d-glucosamine (UDP-GlcNAc) is epimerized at C2 to form UDP-α-N-acetyl-d-mannosamine (UDP-ManNAc). This product is then oxidized by a NAD+-dependent C6-dehydrogenase to form UDP-ManNAcA. In the HS:6 serotype (C. jejuni strain 81116), we identified three enzymes that are required for the biosynthesis of CMP-ß-N-acetyl-d-neuraminic acid (CMP-Neu5Ac). In the first step, UDP-GlcNAc is epimerized at C2 and subsequently hydrolyzed to form N-acetyl-d-mannosamine (ManNAc) with the release of UDP. This product is then condensed with PEP by N-acetyl-d-neuraminate synthase to form N-acetyl-d-neuraminic acid (Neu5Ac). In the final step, CMP-N-acetyl-d-neuraminic acid synthase utilizes CTP to convert this product into CMP-Neu5Ac. A bioinformatic analysis of these five enzymes from C. jejuni serotypes HS:11 and HS:6 identified other bacterial species that can produce UDP-ManNAcA or CMP-Neu5Ac for CPS formation.

Metabolic Engineering of Escherichia coli for High-Titer Biosynthesis of 3'-Sialyllactose.

3'-Sialyllactose (3'-SL) is among the foremost and simplest sialylated breast milk oligosaccharides. In this study, an engineered Escherichia coli for high-titer 3'-SL biosynthesis was developed by introducing a multilevel metabolic engineering strategy, including (1) the introduction of precursor CMP-Neu5Ac synthesis pathway and high-performance α2,3-sialyltransferase (α2,3-SiaT) genes into strain BZ to achieve de novo synthesis of 3'-SL; (2) optimizing the expression of glmS-glmM-glmU involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathways, and constructing a glutamine cycle system, balancing the precursor pools; (3) analysis of critical intermediates and inactivation of competitive pathway genes to redirect carbon flux to 3'-SL biosynthesis; and (4) enhanced catalytic performance of rate-limiting enzyme α2,3-SiaT by RBS screening, protein tag cloning. The final strain BZAPKA14 yielded 9.04 g/L 3'-SL in a shake flask. In a 3 L bioreactor, fed-batch fermentation generated 44.2 g/L 3'-SL, with an overall yield and lactose conversion of 0.53 g/(L h) and 0.55 mol 3'-SL/mol, respectively.

A novel C-domain-dependent inhibition of the rainbow trout CMP-sialic acid synthetase activity by CMP-deaminoneuraminic acid.

The CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by 31P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout.

Enzymatic Synthesis of Sialic Acids in Microfluidics to Overcome Cross-Inhibitions and Substrate Supply Limitations.

Multienzymatic cascade reactions are a powerful strategy for straightforward and highly specific synthesis of complex materials, such as active substances in drugs. Cross-inhibitions and incompatible reaction steps, however, often limit enzymatic activity and thus the conversion. Such limitations occur, e.g., in the enzymatic synthesis of the biologically active sialic acid cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). We addressed this challenge by developing a confinement and compartmentalization concept of hydrogel-immobilized enzymes for improving the efficiency of the enzyme cascade reaction. The three enzymes required for the synthesis of CMP-Neu5Ac, namely, N-acyl-d-glucosamine 2-epimerase (AGE), N-acetylneuraminate lyase (NAL), and CMP-sialic acid synthetase (CSS), were immobilized into bulk hydrogels and microstructured hydrogel-enzyme-dot arrays, which were then integrated into microfluidic devices. To overcome the cytidine triphosphate (CTP) cross-inhibition of AGE and NAL, only a low CTP concentration was applied and continuously conveyed through the device. In a second approach, the enzymes were compartmentalized in separate reaction chambers of the microfluidic device to completely avoid cross-inhibitions and enable the use of higher substrate concentrations. Immobilization efficiencies of up to 25% and pronounced long-term activity of the immobilized enzymes for several weeks were realized. Moreover, immobilized enzymes were less sensitive to inhibition and the substrate-channeling effect between immobilized enzymes promoted the overall conversion in the trienzymatic cascade reaction. Based on this, CMP-Neu5Ac was successfully synthesized by immobilized enzymes in noncompartmentalized and compartmentalized microfluidic devices. This study demonstrates the high potential of immobilizing enzymes in (compartmentalized) microfluidic devices to perform multienzymatic cascade reactions despite cross-inhibitions under continuous flow conditions. Due to the ease of enzyme immobilization in hydrogels, this concept is likely applicable for many cascade reactions with or without cross-inhibition characteristics.

Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro.

A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Inhibition of CMP-sialic acid transport by endogenous 5-methyl CMP.

Nucleotide-sugar transporters (NSTs) transport nucleotide-sugar conjugates into the Golgi lumen where they are then used in the synthesis of glycans. We previously reported crystal structures of a mammalian NST, the CMP-sialic acid transporter (CST) (Ahuja and Whorton 2019). These structures elucidated many aspects of substrate recognition, selectivity, and transport; however, one fundamental unaddressed question is how the transport activity of NSTs might be physiologically regulated as a means to produce the vast diversity of observed glycan structures. Here, we describe the discovery that an endogenous methylated form of cytidine monophosphate (m5CMP) binds and inhibits CST. The presence of m5CMP in cells results from the degradation of RNA that has had its cytosine bases post-transcriptionally methylated through epigenetic processes. Therefore, this work not only demonstrates that m5CMP represents a novel physiological regulator of CST, but it also establishes a link between epigenetic control of gene expression and regulation of glycosylation.

A Phase Ib Open-Label, Dose-Escalation Study of NUC-1031 in Combination with Carboplatin for Recurrent Ovarian Cancer.

PURPOSE: NUC-1031 is a first-in-class ProTide modification of gemcitabine. In PRO-002, NUC-1031 was combined with carboplatin in recurrent ovarian cancer. PATIENTS AND METHODS: NUC-1031 was administered on days 1 and 8 with carboplatin on day 1 every 3 weeks for up to six cycles. Four dose cohorts of NUC-1031 (500, 625, and 750 mg/m2) with carboplatin (AUC4 or 5) were investigated. Primary endpoint was recommended phase II combination dose (RP2CD). Secondary endpoints included safety, investigator-assessed objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), and pharmacokinetics. RESULTS: A total of 25 women with recurrent ovarian cancer, a mean of 3.8 prior lines of chemotherapy, and a median platinum-free interval of 5 months (range: 7-451 days) were enrolled; 15 of 25 (60%) were platinum resistant, 9 (36%) were partially platinum sensitive, and 1 (4%) was platinum sensitive. Of the 23 who were response evaluable, there was 1 confirmed complete response (4%), 5 partial responses (17%), and 8 (35%) stable disease. The ORR was 26% and CBR was 74% across all doses and 100% in the RP2CD cohort. Median PFS was 27.1 weeks. NUC-1031 was stable in the plasma and rapidly generated high intracellular dFdCTP levels that were unaffected by carboplatin. CONCLUSIONS: NUC-1031 combined with carboplatin is well tolerated in recurrent ovarian cancer. Highest efficacy was observed at the RP2CD of 500 mg/m2 NUC-1031 on days 1 and 8 with AUC5 carboplatin day 1, every 3 weeks for six cycles. The ability to deliver carboplatin at AUC5 and the efficacy of this schedule even in patients with platinum-resistant disease makes this an attractive therapeutic combination.

A Phase Ib Study of NUC-1031 in Combination with Cisplatin for the First-Line Treatment of Patients with Advanced Biliary Tract Cancer (ABC-08).

BACKGROUND: Cisplatin/gemcitabine is standard first-line treatment for patients with advanced biliary tract cancer (ABC). NUC-1031 (phosphoramidate transformation of gemcitabine) is designed to enhance efficacy by maximizing intratumoral active metabolites. METHODS: Patients with untreated ABC, Eastern Cooperative Oncology Group performance status 0-1 received NUC-1031 (625 or 725 mg/m2 ) and cisplatin (25 mg/m2 ) on days 1 and 8, every 21 days. Primary objectives were safety and maximum tolerated dose; secondary objectives were objective response rate (ORR), pharmacokinetics, progression-free survival (PFS), and overall survival (OS). RESULTS: Twenty-one patients (median age 61 years, n = 13 male; 17 cholangiocarcinoma, 2 ampullary, and 2 gallbladder cancer) received NUC-1031 625 mg/m2 (n = 8 and expansion n = 7; median six cycles) or 725 mg/m2 (n = 6; median 7.5 cycles). Treatment was well tolerated; most common treatment-emergent grade 3-4 adverse events occurring in more than one patient with 625 mg/m2 NUC-1031 were increased gamma-glutamyl transferase (GGT), 40%; alanine aminotransferase, 20%; bilirubin, 13%; neutropenia, 27%; decreased white cell count, 20%; thrombocytopenia, 13%; nausea, 13%; diarrhea, 13%; fatigue, 13%; and thrombus, 20% and with 725 mg/m2 , increased GGT, 67%, and fatigue, 33%. NUC-1031 725 mg/m2 was selected as the recommended dose with cisplatin in ABC. ORR was 33% (one complete response, six partial responses), DCR was 76%, median PFS was 7.2 months (95% confidence interval [CI], 4.3-10.1), and median OS was 9.6 months (95% CI, 6.7-13.1). The median estimates of area under the plasma concentration-time curve from time 0 to last measurable time and maximum concentration were highest for NUC-1031 (218-324 µg•h/mL and 309-889 µg/mL, respectively) and lowest for di-fluoro-deoxycytidine (0.47-1.56 µg•h/mL and 0.284-0.522 µg/mL, respectively). CONCLUSION: This is the first study reporting on the combination of NUC-1031 with cisplatin in ABC and demonstrated a favorable safety profile; 725 mg/m2 NUC-1031 in combination with cisplatin is undergoing phase III trial evaluation in ABC. (ClinicalTrials.gov ID: NCT02351765; EudraCT ID: 2015-000100-26). IMPLICATIONS FOR PRACTICE: The prognosis for patients with advanced biliary tract cancer (ABC) is approximately 1 year, and new treatment options are required. The cisplatin/gemcitabine combination is standard first-line treatment for patients with ABC. NUC-1031 is a phosphoramidate transformation of gemcitabine and is designed to enhance efficacy by maximizing intratumoral active metabolites. This phase Ib study (ABC-08) demonstrated a favorable safety profile of NUC-1031 in combination with cisplatin for the first-line treatment of patients with ABC, and 725 mg/m2 NUC-1031 was recommended in combination with cisplatin for phase III trial evaluation; the NuTide:121 global randomized study is currently enrolling.

Unliganded and CMP-Neu5Ac bound structures of human α-2,6-sialyltransferase ST6Gal I at high resolution.

Sialic acid residues found as terminal monosaccharides in various types of glycan chains in cell surface glycoproteins and glycolipids have been identified as important contributors of cell-cell interactions in normal vs. abnormal cellular behavior and are pivotal in diseases such as cancers. In vertebrates, sialic acids are attached to glycan chains by a conserved subset of sialyltransferases with different enzymatic and substrate specificities. ST6Gal I is a sialyltransferase using activated CMP-sialic acids as donor substrates to catalyze the formation of a α2,6-glycosidic bond between the sialic acid residue and the acceptor disaccharide LacNAc. Understanding sialyltransferases at the molecular and structural level shed light into their function. We present here two human ST6Gal I structures, which show for the first time the enzyme in the unliganded state and with the full donor substrate CMP-Neu5Ac bound. Comparison of these structures reveal flexibility of the catalytic loop, since in the unliganded structure Tyr354 adopts a conformation seen also as an alternate conformation in the substrate bound structure. CMP-Neu5Ac is bound with the side chain at C5 of the sugar residue directed outwards at the surface of the protein. Furthermore, the exact binding mode of the sialic acid moiety of the substrate directly involves sialylmotifs L, S and III and positions the sialylmotif VS in the immediate vicinity. We also present a model for the ternary complex of ST6Gal I with both the donor and the acceptor substrates.

Gram-Negative Bacterial Endotoxin LPS Induces NeuGc Loss through Ets1-Dependent Downregulation of Intestine-Specific pcmah Transcript in Porcine Intestinal Cells.

N-glycolylneuraminic acid (NeuGc), a non-human sialic acid derivative synthesized by cytidine-5'-monophospho-N-acetylneuraminic acid hydroxylase (CMAH), plays a crucial role in mediating infections by certain pathogens. Although it has been postulated that NeuGc biosynthesis and CMAH expression are downregulated during microbial infection, the underlying mechanisms remain unclear. The present study showed that exposure to lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, leads to loss of NeuGc biosynthesis in pig small intestinal I2I-2I cells. This LPS-induced NeuGc loss was accompanied by decreased CMAH transcript levels, especially intestine-specific 5'pcmah-1. Furthermore, LPS suppressed the activity of the Pi promoter responsible for 5'pcmah-1 by inhibiting DNA binding of Est1. These findings provide insight into the regulatory mechanisms of Neu5Gc biosynthesis during pathogenic infectious events, which may represent a host defense mechanism that protects the self against pathogenic bacterial infections even in non-sanitary environments.

Efficacy of Antigonococcal CMP-Nonulosonate Therapeutics Require Cathelicidins.

Novel therapies to counteract multidrug-resistant gonorrhea are urgently needed. A unique gonococcal immune evasion strategy involves capping of lipooligosaccharide (LOS) with sialic acid by gonococcal sialyltransferase (Lst), utilizing host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation renders gonococci resistant to complement and cationic peptides, and down-regulates the inflammatory response by engaging siglecs. CMP-sialic acid analogs (CMP-nonulosonates [CMP-NulOs]) such as CMP-Leg5,7Ac2 and CMP-Kdn are also utilized by Lst. Incorporation of these NulO analogs into LOS maintains gonococci susceptible to complement. Intravaginal administration of CMP-Kdn or CMP-Leg5,7Ac2 attenuates gonococcal colonization of mouse vaginas. Here, we identify a key mechanism of action for the efficacy of CMP-NulOs. Surprisingly, CMP-NulOs remained effective in complement C1q-/- and C3-/- mice. LOS Neu5Ac, but not Leg5,7Ac2 or Kdn, conferred resistance to the cathelicidins LL-37 (human) and mouse cathelicidin-related antimicrobial peptide in vitro. CMP-NulOs were ineffective in Camp-/- mice, revealing that cathelicidins largely mediate the efficacy of therapeutic CMP-NulOs.

NUC-1031, use of ProTide technology to circumvent gemcitabine resistance: current status in clinical trials.

BACKGROUND: Resistance to gemcitabine chemotherapy is common in patients with pancreatic ductal adenocarcinoma (PDAC), biliary tract cancer (BTC) and ovarian cancers (OC), conferring poor survival. Use of ProTide technology led to the development of a 'partially-activated' monophosphorylated gemcitabine compound, termed NUC-1031. NUC-1031 enters cancer cells independent of the human equilibrative nucleoside transporter, does not require deoxycytidine kinase-mediated activation and resists cytidine deaminase-mediated breakdown into toxic by-products. CURRENT FINDINGS: The phase I PRO-001 trial recruited 68 patients with advanced solid tumours; of the 49 patients that had response-evaluable disease, 5 (10%) had a partial response (PR) and 33 (67%) had stable disease (SD). Subsequently, the PRO-002 study assessed the safety and efficacy of NUC-1031 combined with carboplatin for patients with OC (n = 25); preliminary data from this study reported one (4%) unconfirmed complete response (CR), 8 (35%) PRs and 13 (57%) patients with SD, the final outcome data are awaited. The ABC-08 trial for advanced BTC assessed safety and efficacy of NUC-1031 combined with cisplatin; 14 patients were recruited with a 50% objective response rate in the intention to treat population at interim analysis. ACELARATE, the phase III trial in first-line advanced PDAC comparing NUC-1031 to gemcitabine monotherapy, recruited 200 patients but has been paused for futility analysis. CONCLUSION: Early studies demonstrate NUC-1031 is well tolerated with favourable pharmacokinetic profiles. NUC-1031 use in PDAC remains unclear, but encouraging results of disease control in BTC and OC has prompted phase II and III trial development. NuTide 121, is a phase III trial comparing cisplatin-NUC 1031 combination to the standard of care cisplatin-gemcitabine and recruitment is ongoing. Recruiting trials and mature data from existing studies will help inform on the impact of NUC-1031 on patient survival over standard gemcitabine.

NUC-1031 in biliary tract cancer: from bench to bedside and back?

In preclinical models of biliary tract cancer, NUC-1031 showed less potency than gemcitabine, no correlation with potential biomarkers and only moderate additive interaction in combination with cisplatin. These findings should prompt further careful pharmacological and translational studies to better define the purported therapeutic advantage of NUC-1031 over gemcitabine. That would be a more cautious approach than the phase III clinical trial which is planning to enrol 828 patients with biliary tract tumours to compare gemcitabine/cisplatin "conventional" treatment with or without NUC-1031.

NUC-1031/cisplatin versus gemcitabine/cisplatin in untreated locally advanced/metastatic biliary tract cancer (NuTide:121).

Gemcitabine/cisplatin is standard of care for first-line treatment of patients with advanced biliary tract cancer (aBTC); new treatments are needed. NUC-1031 is designed to overcome key cancer resistance mechanisms associated with gemcitabine. The tolerability/efficacy signal of NUC-1031/cisplatin in the Phase Ib ABC-08 study suggested that this combination may represent a more efficacious therapy than gemcitabine/cisplatin for patients with aBTC, leading to initiation of the global NuTide:121 study which will include 828 patients ≥18 years with untreated histologically/cytologically-confirmed aBTC (including cholangiocarcinoma, gallbladder or ampullary cancer); randomized (1:1) to NUC-1031 (725 mg/m2)/cisplatin (25 mg/m2) or gemcitabine (1000 mg/m2)/cisplatin (25 mg/m2), on days 1/8, Q21-days. Primary objectives are overall survival and objective response rate. Secondary objectives: progression-free survival, safety, pharmacokinetics, patient-reported quality of life and correlative studies. (Investigational new drug (IND) number: 139058, European Clinical Trials database: EudraCT Number 2019-001025-28, ClinicalTrials.gov identifier: NCT04163900).

Therapeutic CMP-Nonulosonates against Multidrug-Resistant Neisseria gonorrhoeae.

Neisseria gonorrhoeae deploys a unique immune evasion strategy wherein the lacto-N-neotetraose termini of lipooligosaccharide (LOS) are "capped" by a surface LOS sialyltransferase (Lst), using extracellular host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation enhances complement resistance by recruiting factor H (FH; alternative complement pathway inhibitor) and also by limiting classical pathway activation. Sialylated LOS also engages inhibitory Siglecs on host leukocytes, dampening innate immunity. Previously, we showed that analogues of CMP-sialic acids (CMP-nonulosonates [CMP-NulOs]), such as CMP-Leg5,7Ac2 and CMP-Neu5Ac9N3, are also substrates for Lst. Incorporation of Leg5,7Ac2 and Neu5Ac9N3 into LOS results in N. gonorrhoeae being fully serum sensitive. Importantly, intravaginal administration of CMP-Leg5,7Ac2 attenuated N. gonorrhoeae colonization of mouse vaginas. In this study, we characterize and develop additional candidate therapeutic CMP-NulOs. CMP-ketodeoxynonulosonate (CMP-Kdn) and CMP-Kdn7N3, but not CMP-Neu4,5Ac2, were substrates for Lst, further elucidating gonococcal Lst specificity. Lacto-N-neotetraose LOS capped with Kdn and Kdn7N3 bound FH to levels ∼60% of that seen with Neu5Ac and enabled gonococci to resist low (3.3%) but not higher (10%) concentrations of human complement. CMP-Kdn, CMP-Neu5Ac9N3, and CMP-Leg5,7Ac2 administered intravaginally (10 µg/d) to N. gonorrhoeae-colonized mice were equally efficacious. Of the three CMP-NulOs above, CMP-Leg5,7Ac2 was the most pH and temperature stable. In addition, Leg5,7Ac2-fed human cells did not display this NulO on their surface. Moreover, CMP-Leg5,7Ac2 was efficacious against several multidrug-resistant gonococci in mice with a humanized sialome (Cmah-/- mice) or humanized complement system (FH/C4b-binding protein transgenic mice). CMP-Leg5,7Ac2 and CMP-Kdn remain viable leads as topical preventive/therapeutic agents against the global threat of multidrug-resistant N. gonorrhoeae.

Evaluation of NUC-1031: a first-in-class ProTide in biliary tract cancer.

PURPOSE: NUC1031 is a first-in-class ProTide, that is a gemcitabine pro-drug designed to overcome putative mechanisms of resistance, including decreased expression of hENT/hCNT transporters, absence of activating enzymes such as deoxycytidine kinase (dCK) and presence of degrading enzymes such as cytidine deaminase (CDA). We undertook comprehensive pre-clinical evaluation of NUC1031 in biliary tract cancer (BTC) models, given that gemcitabine/cisplatin is a standard first-line therapy in advanced BTC. METHODS: Here, we compared the in vitro activity of NUC1031 in comparison to gemcitabine, validate putative mechanism(s) of action, assessed potential biomarkers of sensitivity or resistance, and performed combination studies with cisplatin. We also evaluated the in vivo efficacy of NUC1031 and gemcitabine using a CDA-high cholangiocarcinoma patient-derived xenograft (PDX) model. RESULTS: In a panel of BTC cell lines (N = 10), NUC1031 had less potency than gemcitabine in multiple cellular assays. NUC1031 did not demonstrate evidence of greater synergy over gemcitabine in combination with cisplatin. Surprisingly, efficacy of both gemcitabine and NUC1031 was not found to be correlated with hENT/hCTN, dCK or CDA transcript levels. Gemcitabine and NUC1031 showed equivalent efficacy in a CDA-high PDX model in vivo contradicting the primary rationale of NUC1031 design. CONCLUSION: NUC1031 did not exhibit evidence of superior activity over gemcitabine, as a single-agent, or in combination with cisplatin, in either our in vivo or in vitro BTC models. Given that the largest Phase 3 study (ClinicalTrials.gov: NCT0314666) to date in BTC is underway (N = 828) comparing NUC1031/cisplatin to gemcitabine/cisplatin, our results suggest that a more conservative clinical evaluation path would be more appropriate.

MeCP2 epigenetically regulates alpha-smooth muscle actin in human lung fibroblasts.

BACKGROUND: A critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (α-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating α-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in α-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of α-SMA expression. METHODS: The association of MeCP2 with α-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor ß1 (TGF-ß1). The regulation of α-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on α-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of α-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing. RESULTS: The human lung fibroblasts with increased α-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced α-SMA upregulation by TGF-ß1. The 5'-Aza-treated cells have decreased α-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of α-SMA after being treated by TGF-ß1 or 5'-aza. CONCLUSION: Our data indicate that the methyl-binding protein MeCP2 is critical for α-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of α-SMA is not a determinative factor for its inducible expression.

Quantitation of cytidine-5'-monophospho-N-acetylneuraminic acid in human leukocytes using LC-MS/MS: method development and validation.

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.