Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria-causing parasites.

The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target.

Synthesis of 5'-Thymidine-Conjugated Formylphenylboronic Acids as Potential Lysine Targeting Iminoboronate Reversible Covalent Enzyme Probes.

The design of reversible-covalent molecules to selectively target the ε-amino functionality of lysine residues in enzymes or proteins is a highly desirable goal. Herein, we describe synthetic methodology used to prepare a series of 5'-thymidine-linked formylphenylboronic acids as probes to interrogate sugar nucleotide processing enzymes that recognize thymidine. The first synthetic strategy mitigated the need for protecting group manipulations of thymidine by capitalizing upon the straightforward preparation, isolation, and reactivity of 5'-azidothymidine. An alkyne cycloaddition partner was installed through either a propargyl or ethynyl phenyl ketone derived boronic acid. The second strategy directly linked formylphenylboronic acids to 5-thymidine through an ether linkage installed using Mitsunobu conditions with 3'-O,3-dibenzoylthymidine. Iminoboronate formation was observed with a selected probe.

Pilot study of jadomycin B pharmacokinetics and anti-tumoral effects in zebrafish larvae and mouse breast cancer xenograft models.

Despite numerous therapeutic options, multidrug resistance (MDR) remains an obstacle to successful breast cancer therapy. Jadomycin B, a natural product derived from Streptomyces venezuelae ISP5230, maintains cytotoxicity in MDR human breast cancer cells. Our objectives were to evaluate the pharmacokinetics, toxicity, anti-tumoral, and anti-metastatic effects of jadomycin B in zebrafish larvae and mice. In a zebrafish larval xenograft model, jadomycin B significantly reduced the proliferation of human MDA-MB-231 cells at or below its maximum tolerated dose (40 µm). In female Balb/C mice, a single intraperitoneal dose (6 mg/kg) was rapidly absorbed with a maximum serum concentration of 3.4 ± 0.27 µm. Jadomycin B concentrations declined biphasically with an elimination half-life of 1.7 ± 0.058 h. In the 4T1 mouse mammary carcinoma model, jadomycin B (12 mg/kg every 12 h from day 6 to 15 after tumor cell injection) decreased primary tumor volume compared to vehicle control. Jadomycin B-treated mice did not exhibit weight loss, nor significant increases in biomarkers of impaired hepatic (alanine aminotransferase) and renal (creatinine) function. In conclusion, jadomycin B demonstrated a good safety profile and provided partial anti-tumoral effects, warranting further dose-escalation safety and efficacy studies in MDR breast cancer models.

5'-Chalcogen-Substituted Nucleoside Pyrophosphate and Phosphate Monoester Analogues: Preparation and Hydrolysis Studies.

Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using 31P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.

A missense variant remote from the active site impairs stability of human phosphoglucomutase 1.

Missense variants of human phosphoglucomutase 1 (PGM1) cause the inherited metabolic disease known as PGM1 deficiency. This condition is categorised as both a glycogen storage disease and a congenital disorder of glycosylation. Approximately 20 missense variants of PGM1 are linked to PGM1 deficiency, and biochemical studies have suggested that they fall into two general categories: those affecting the active site and catalytic efficiency, and those that appear to impair protein folding and/or stability. In this study, we characterise a novel variant of Arg422, a residue distal from the active site of PGM1 and the site of a previously identified disease-related variant (Arg422Trp). In prior studies, the R422W variant was found to produce insoluble protein in a recombinant expression system, precluding further in vitro characterisation. Here we investigate an alternative variant of this residue, Arg422Gln, which is amenable to experimental characterisation presumably due to its more conservative physicochemical substitution. Biochemical, crystallographic, and computational studies of R422Q establish that this variant causes only minor changes in catalytic efficiency and 3D structure, but is nonetheless dramatically reduced in stability. Unexpectedly, binding of a substrate analog is found to further destabilise the protein, in contrast to its stabilising effect on wild-type PGM1 and several other missense variants. This work establishes Arg422 as a lynchpin residue for the stability of PGM1 and supports the impairment of protein stability as a pathomechanism for variants that cause PGM1 deficiency. SYNOPSIS: Biochemical and structural studies of a missense variant far from the active site of human PGM1 identify a residue with a key role in enzyme stability.

On the Catalytic Activity of a GT1 Family Glycosyltransferase from Streptomyces venezuelae ISP5230.

GT1 family glycosyltansferase, Sv0189, from Streptomyces venezuelae ISP5230 (ATCC 10721) was characterized. The recombinantly produced protein Sv0189 possessed UDP-glycosyltransferase activity. Screening, using an assay employing unnatural nitrophenyl glycosides as activated donors, resulted in the discovery of a broad substrate scope with respect to both acceptor molecules and donor sugars. In addition to polyphenols, including anthraquinones, simple aromatics containing primary or secondary alcohols, a variety of complex natural products and synthetic drugs were glucosylated or xylosylated by Sv0189. Regioselectivity was established through the isolation and characterization of glucosylated products. Sv0189 and homologous proteins are widely distributed among Streptomyces species, and their apparent substrate promiscuity reveals potential for their development as biocatalysts for glycodiversification.

Inhibitory Evaluation of αPMM/PGM from Pseudomonas aeruginosa: Chemical Synthesis, Enzyme Kinetics, and Protein Crystallographic Study.

α-Phosphomannomutase/phosphoglucomutase (αPMM/PGM) from P. aeruginosa is involved in bacterial cell wall assembly and is implicated in P. aeruginosa virulence, yet few studies have addressed αPMM/PGM inhibition from this important Gram-negative bacterial human pathogen. Four structurally different α-d-glucopyranose 1-phosphate (αG1P) derivatives including 1-C-fluoromethylated analogues (1-3), 1,2-cyclic phosph(on)ate analogues (4-6), isosteric methylene phosphono analogues (7 and 8), and 6-fluoro-αG1P (9), were synthesized and assessed as potential time-dependent or reversible αPMM/PGM inhibitors. The resulting kinetic data were consistent with the crystallographic structures of the highly homologous Xanthomonas citri αPGM with inhibitors 3 and 7-9 binding to the enzyme active site (1.65-1.9 Å). These structural and kinetic insights will enhance the design of future αPMM/PGM inhibitors.

Post Polyketide Synthase Carbon-Carbon Bond Formation in Type-II PKS-Derived Natural Products from Streptomyces venezuelae.

Polyketide synthase (PKS) derived natural products are biosynthesized by head-to-tail addition of acetate and malonate extender units resulting in linear extended-polyketide chains. Despite the well-documented structural diversity associated with PKS-derived natural products, C-C chain branching deviating from the usual linear pattern is relatively rare. Herein, type-II PKS angucyclic natural products containing a hemiaminal functionality were identified and proposed as the parent of a series of C-C-branched analogues. These C-C linked acetate or pyruvate branching units were located at the α-positions on the extended polyketide chains of jadomycins incorporating 3- and 4-aminomethylbenzoic acids. Labeling studies utilizing [1-13C]-d-glucose provided mechanistic evidence that the C-C bond formation occurred as a result of a previously unidentified post-PKS processing, additional to the enzymes encoded within the biosynthetic gene cluster. Selected compounds were evaluated in cytotoxic or antimicrobial assays.

MgF3- and AlF4- transition state analogue complexes of yeast phosphoglycerate kinase.

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) transition state analogue complexes and analyzed using 19F, 1H waterLOGSY and 1H chemical shift perturbation NMR spectroscopy. We observed the first 19F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.

Jadomycins Inhibit Type II Topoisomerases and Promote DNA Damage and Apoptosis in Multidrug-Resistant Triple-Negative Breast Cancer Cells.

Jadomycins are natural products that kill drug-sensitive and multidrug-resistant (MDR) breast cancer cells. To date, the cytotoxic activity of jadomycins has never been tested in MDR breast cancer cells that are also triple negative. Additionally, there is only a rudimentary understanding of how jadomycins cause cancer cell death, which includes the induction of intracellular reactive oxygen species (ROS). We first created a paclitaxel-resistant, triple-negative breast cancer cell line [paclitaxel-resistant MDA-MB-231 breast cancer cells (231-TXL)] from drug-sensitive control MDA-MB-231 cells (231-CON). Using thiazolyl blue methyltetrazolium bromide cell viability-measuring assays, jadomycins B, S, and F were found to be equipotent in drug-sensitive 231-CON and MDR 231-TXL cells; and using ROS-detecting assays, these jadomycins were determined to increase ROS activity in both cell lines by up to 7.3-fold. Jadomycins caused DNA double-strand breaks in 231-CON and 231-TXL cells as measured by γH2AX Western blotting. Coincubation with the antioxidant N-acetyl cysteine or pro-oxidant auranofin did not affect jadomycin-mediated DNA damage. Jadomycins induced apoptosis in 231-CON and 231-TXL cells as measured by annexin V affinity assays, a process that was retained when ROS were inhibited. This indicated that jadomycins are capable of inducing MDA-MB-231 apoptotic cell death independently of ROS activity. Using quantitative polymerase chain reaction, Western blotting, and direct topoisomerase inhibition assays, it was determined that jadomycins inhibit type II topoisomerases and that jadomycins B and F selectively poison topoisomerase IIß We therefore propose novel mechanisms through which jadomycins induce breast cancer cell death independently of ROS activity, through inhibition or poisoning of type II topoisomerases and the induction of DNA damage and apoptosis.

Furan and Lactam Jadomycin Biosynthetic Congeners Isolated from Streptomyces venezuelae ISP5230 Cultured with Nε-Trifluoroacetyl-l-lysine.

Angucycline antibiotics are composed of a classical four-ring angularly linked polyaromatic backbone. Differential cyclization chemistry of the A- and B-rings in jadomycin biosynthesis led to the discovery of two new furan analogues, while oxidation led to a ring-opened form of the jadomycin Nε-trifluoroacetyl-l-lysine (TFAL) congener. The compounds were isolated from Streptomyces venezuelae ISP5230 cultures grown with TFAL. Biosynthetic incorporation using d-[1-13C]-glucose in cultures enabled the unambiguous assignment of the aldehyde, alcohol, and amide functionalities present in these new congeners through NMR spectroscopy. Tandem mass spectrometry analysis of cultures grown with 15Nα- or 15Nε-lysine demonstrated the incorporation of Nα exclusively into the angucycline backbone, contrasting results with ornithine [J. Am. Chem. Soc. 2015, 137, 3271]. Compounds were evaluated against antimicrobial and cancer cell panels and found to possess good activity against Gram-positive bacteria.

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction.

ß-Phosphoglucomutase (ßPGM) catalyzes isomerization of ß-D-glucose 1-phosphate (ßG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a ß-D-glucose 1,6-bisphosphate (ßG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of ßG1P deliver novel step 1 transition state analog (TSA) complexes for ßPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the ß-D-glucopyranose ring in the ßG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of ßG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.

JadX is a Disparate Natural Product Binding Protein.

We report that JadX, a protein of previously undetermined function coded for in the jadomycin biosynthetic gene cluster of Streptomyces venezuelae ISP5230, affects both chloramphenicol and jadomycin production levels in blocked mutants. Characterization of recombinant JadX through protein-ligand interactions by chemical shift perturbation and WaterLOGSY NMR spectroscopy resulted in the observation of binding between JadX and a series of jadomycins and between JadX and chloramphenicol, another natural product produced by S. venezuelae ISP5230. These results suggest JadX to be an unusual class of natural product binding protein involved in binding structurally disparate natural products. The ability for JadX to bind two different natural products in vitro and the ability to affect production of these secondary metabolites in vivo suggest a potential role in regulation or signaling. This is the first example of functional characterization of these JadX-like proteins, and provides insight into a previously unobserved regulatory process.

Synthesis of α-Deoxymono and Difluorohexopyranosyl 1-Phosphates and Kinetic Evaluation with Thymidylyl- and Guanidylyltransferases.

Eight fluorinated isosteric α-d-glucopyranosyl 1-phosphate (Glc 1P) analogues have been synthesized. A promiscuity investigation of the thymidylyltransferase Cps2L and the guanidylyltansferase GDP-ManPP with these analogues showed that all were accepted by either enzyme, with the exception of 1,6-diphosphate 6. Kinetic parameters were determined for these analogues using a continuous coupled assay. These data demonstrated the broad substrate promiscuity of Cps2L, with kcat/Km changes for monofluoro substitution at C-2, C-4, and C-6 and difluoro substitution at C-2 within two orders of magnitude. In contrast, the kinetic analysis of GDP-ManPP was only possible with three out of eight analogues. The pKa2 values of analogues (1-3) were determined by proton decoupled 31P and 19F NMR titration experiments. Counterintuitively, the axial fluoro substituent in 3 did not change chemical shift upon titration, and there was no significant increase in acidity for the difluoro analogue over the monofluoro analogues. No strong Brønsted linear free-energy correlations were observed among all five substrates (1-3, Glc 1P, and Man 1P) for either enzyme-catalyzed reactions. However, Brønsted correlations were observed among selected substrates, indicating that the acidity of the nucleophilic phosphate and the configuration of the hexose each plays a significant role in determining the substrate specificity.

Mechanistic evaluation of a nucleoside tetraphosphate with a thymidylyltransferase.

Pyrimidine polyphosphates were first detected in cells 5 decades ago; however, their biological significance remains only partially resolved. Such nucleoside polyphosphates are believed to be produced nonspecifically by promiscuous enzymes. Herein, synthetically prepared deoxythymidine 5'-tetraphosphate (p4dT) was evaluated with a thymidylyltransferase, Cps2L. We have identified p4dT as a substrate for Cps2L and evaluated the reaction pathway by analysis of products using high-performance liquid chromatography, liquid chromatography and tandem mass spectrometry, and 31P nuclear magnetic resonance spectroscopy. Product analysis confirmed production of dTDP-Glc and triphosphate (P3) and showed no trace of dTTP-Glc and PPi, which could arise from alternative pathways for the reaction mechanism.

Eight-membered ring-containing jadomycins: implications for non-enzymatic natural products biosynthesis.

Jadomycin Oct (1) was isolated from Streptomyces venezuelae ISP5230 and characterized as a structurally unique eight-membered l-ornithine ring-containing jadomycin. The structure was elucidated through the semisynthetic derivatization of starting material via chemoselective acylation of the l-ornithine α-amino group using activated succinimidyl esters. Incorporation of 5-aminovaleric acid led to jadomycin AVA, a second eight-membered ring-containing jadomycin. These natural products illustrate the structural diversity permissible from a non-enzymatic step within a biosynthetic pathway and exemplifies the potential for discovery of novel scaffolds.

On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer.

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

Polyphosphate-containing bisubstrate analogues as inhibitors of a bacterial cell wall thymidylyltransferase.

A series of polyphosphate containing sugar nucleotide analogues were synthesized and evaluated as bisubstrate inhibitors of α-D-glucose 1-phosphate thymidylyltransferase Cps2L, the first enzyme in Streptococcus pneumoniael-rhamnose biosynthesis, and a novel antibacterial target. WaterLOGSY NMR spectroscopy demonstrated binding of bisubstrate analogues to Cps2L and a spectrophotometric coupled assay was used to determine apparent Ki values.

Investigations into the binding of jadomycin DS to human topoisomerase IIß by WaterLOGSY NMR spectroscopy.

The jadomycins are a family of secondary metabolites produced by S. venezuelae ISP5230. Specific jadomycins have been shown to possess a variety of anticancer, antifungal, and antibacterial properties, with different molecular mechanisms of action. Herein we demonstrate qualitative and quantitative direct binding between the validated anticancer target human topoisomerase IIß and jadomycin DS using WaterLOGSY NMR spectroscopy. Additionally, we report for the first time, that jadomycin DS also binds a variety of other proteins, likely in a non-specific manner. Such interactions may rationalize the potential polypharmacology of jadomycin DS.