Optimized plant compound with potent anti-biofilm activity across gram-negative species.

Many human diseases, including cystic fibrosis lung infections, are caused or exacerbated by bacterial biofilms. Specialized modes of motility, including swarming and twitching, allow gram-negative bacteria to spread across surfaces and form biofilms. Compounds that inhibit these motilities could slow the spread of biofilms, thereby allowing antibiotics to work better. We previously demonstrated that a set of plant-derived triterpenes, including oleanolic acid and ursolic acid, inhibit formation of Escherichia coli and Pseudomonas aeruginosa biofilms, and alter expression of genes involved in chemotaxis and motility. In the present study, we have prepared a series of analogs of oleanolic acid. The analogs were evaluated against clinical isolates of E. coli and P. aeruginosa in biofilm formation assays and swarming assays. From these analogs, compound 9 was selected as a lead compound for further development. Compound 9 inhibits E. coli biofilm formation at 4 µg/mL; it also inhibits swarming at ≤1 µg/mL across multiple clinical isolates of P. aeruginosa, E. coli, Burkholderia cepacia, and Salmonella enterica, and at <0.5 µg/mL against multiple agricultural strains. Compound 9 also potentiates the activity of the antibiotics tobramycin and colistin against swarming P. aeruginosa; this is notable, as tobramycin and colistin are inhaled antibiotics commonly used to treat P. aeruginosa lung infections in people with cystic fibrosis. qPCR experiments suggested that 9 alters expression of genes involved in regulating Type IV pili; western blots confirmed that expression of Type IV pili components PilA and PilY1 decreases in P. aeruginosa in the presence of 9.

SQ1274, a novel microtubule inhibitor, inhibits ovarian and uterine cancer cell growth.

OBJECTIVE: Paclitaxel, a microtubule inhibitor, is subject to tumor resistance while treating high-grade serous ovarian and uterine cancer. This study aims to directly compare the effects of SQ1274, a novel microtubule inhibitor that binds to the colchicine-binding site on tubulin, and paclitaxel in high-grade serous ovarian and uterine cancer cell lines both in vitro and in vivo. METHODS: We assessed the sensitivity of ovarian (OVCAR8) and uterine (ARK1) cancer cell lines to SQ1274 and paclitaxel using XTT assays. We used western blot and quantitative real-time PCR to analyze changes in AXL RNA and protein expression by SQ1274 and paclitaxel. Differences in cell-cycle arrest and apoptosis were investigated using flow cytometry. Finally, we treated ovarian and uterine xenograft models with vehicle, paclitaxel, or SQ1274. RESULTS: First, we demonstrate that SQ1274 has a much lower IC50 than paclitaxel in both ARK1 (1.26 nM vs. 15.34 nM, respectively) and OVCAR8 (1.34 nM vs. 10.29 nM, respectively) cancer cell lines. Second, we show SQ1274 decreases both RNA and protein expression of AXL. Third, we show that SQ1274 causes increased cell-cycle arrest and apoptosis compared to paclitaxel. Finally, we report that SQ1274 more effectively inhibits tumor growth in vivo compared to paclitaxel. CONCLUSIONS: SQ1274 presents as a viable alternative to paclitaxel for treating ovarian and uterine cancer. This study supports the development of SQ1274 as a chemotherapeutic to treat ovarian and uterine cancer.

Evolution of allosteric regulation in chorismate mutases from early plants.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 ŠX-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.

A Total Synthesis of Bifidenone.

The first total synthesis of bifidenone, a novel natural tubulin polymerization inhibitor, has been achieved in 12 steps starting from commercially available 1,4-dioxaspiro[4.5]decan-8-one. The synthesis includes a newly developed method to generate the dihydrobenzodioxolone core by palladium-catalyzed aerobic dehydrogenation. The three stereocenters were installed with an AD-mix-ß dihydroxylation step followed by a late-stage palladium-catalyzed decarboxylation-allylation procedure. The absolute stereochemistry of 3 was determined via 13a by single-crystal X-ray analysis.

Isolation and Identification of the Novel Tubulin Polymerization Inhibitor Bifidenone.

The pursuit of structurally novel compounds has led to the isolation of a series of neolignans (2-6), for which the structures have been determined from microgram quantities using microcryoprobe NMR technology. Compounds 2-6 provided some unexpectedly clear structure-activity relationship data, with compound 2 demonstrating significantly more potency in the in vitro cytotoxicity assay than the other analogues. Further screening found that compound 2 induces apoptosis with activation of caspase 3/7. The NCI Compare algorithm suggested that compound 2 acts through the inhibition of tubulin/microtubule dynamics. Compound 2 was confirmed to be a tubulin polymerization inhibitor that binds directly to tubulin.

Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.

Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells.

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

Digging Deep for New Compounds from the Compass Plant, Silphium laciniatum.

The compass plant, Silphium laciniatum, is an iconic perennial plant of the North American tallgrass prairie. The plants of the tallgrass prairie historically have been subjected to a number of biological and environmental stresses. Among the adaptations developed by S. laciniatum is a large deep taproot. An investigation of the secondary metabolites found in the root of a S. laciniatum specimen has led to the identification of 15 new terpenoids (3-8, 10-17, and 22), which were screened for cytotoxic activity in the NCI-H460 human large-cell lung carcinoma cell line.

Diterpenes from the endangered goldenrod Solidago shortii.

Species extinction is tantamount to loss of chemical diversity, and so it is important to seize all opportunities to study species on the brink of extinction. Such studies are often hampered by the limited material available, but that obstacle is surmountable through collaboration with botanical gardens and advances in instrumentation. The goldenrod Solidago shortii is one example of an endangered species native to the United States. From S. shortii, one known diterpene (1), two new diterpenes (2 and 3), and three new hydrolysis products (4-6) are described. This work was made possible through collaboration with the Missouri Botanical Garden and with the use of highly sensitive microcryoprobe NMR technology for structure elucidation and VCD spectroscopy for the determination of absolute configuration.

Acetylated dammarane-type bisdesmosides from Combretum inflatum.

The first study of the chemical constituents of Combretum inflatum has resulted in the isolation of seven new acetylated dammarane-type bisdesmosides (1-7). Their structures were determined from microgram quantities on hand using Bruker BioSpin TCI 1.7 mm MicroCryoProbe technology, ESIMS, and comparison to data found in the literature. Compounds 1-7 were screened for inhibition of an Escherichia coli strain UTI89 biofilm, MRSA inhibition, and cytotoxicity in NCI-H460 human lung cancer cells. Compounds 3-7 reduced the growth of MRSA at 16 µg/mL by 71-45%, and compound 7 had an IC50 value of 3.9 µM in NCI-H460.

Cytotoxic and antibacterial beilschmiedic acids from a Gabonese species of Beilschmiedia.

High-throughput natural products chemistry methods have facilitated the isolation of eight new (1-8) and two known (9 and 10) beilschmiedic acid derivatives from the leaves of a Gabonese species of Beilschmiedia. Compounds 3-10 were isolated in microgram quantities, and the NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7 mm MicroCryoProbe. All of the compounds were screened for cytotoxic and antibacterial activity against NCI-H460 human lung cancer cells and a clinical isolate of methicillin-resistant Staphylococcus aureus, respectively. This is the first report of cytotoxic activity for the endiandric/beilschmiedic acid class of compounds.

Isolation of apoptosis-inducing stilbenoids from four members of the Orchidaceae family.

High-throughput natural product research produced a suite of anticancer hits among several species of the Orchidaceae family (Oncidium microchilum, O. isthmi, and Myrmecophila humboldtii). A commercial Oncidium sp. was also examined as a convenient source of additional material. Isolation and structure elucidation led to the identification of fifteen stilbenoids including a new phenanthraquinone and two new dihydrostilbenes. NMR data for structure elucidation and dereplication were acquired utilizing a Bruker BioSpin TCI 1.7-mm MicroCryoProbe or a 5-µL CapNMR capillary microcoil. Several compounds inhibited proliferation of NCI-H460 and M14 cancer cell lines. All compounds were also examined for their ability to induce apoptosis. Apoptosis induction was determined by measuring caspase 3/7 activation and LDH release in a NCI-H460 cell line. Based on these results, a portion of the extract from a commercially available Oncidium sp. was chemically modified in an attempt to obtain additional phenanthraquinones.

Treponema denticola PurE Is a bacterial AIR carboxylase.

De novo purine biosynthesis proceeds by two divergent paths. In bacteria, yeasts, and plants, 5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxy-AIR (CAIR) by two enzymes: N(5)-carboxy-AIR (N(5)-CAIR) synthetase (PurK) and N(5)-CAIR mutase (class I PurE). In animals, the conversion of AIR to CAIR requires a single enzyme, AIR carboxylase (class II PurE). The CAIR carboxylate derives from bicarbonate or CO(2), respectively. Class I PurE is a promising antimicrobial target. Class I and class II PurEs are mechanistically related but bind different substrates. The spirochete dental pathogen Treponema denticola lacks a purK gene and contains a class II purE gene, the hallmarks of CO(2)-dependent CAIR synthesis. We demonstrate that T. denticola PurE (TdPurE) is AIR carboxylase, the first example of a prokaryotic class II PurE. Steady-state and pre-steady-state experiments show that TdPurE binds AIR and CO(2) but not N(5)-CAIR. Crystal structures of TdPurE alone and in complex with AIR show a conformational change in the key active site His40 residue that is not observed for class I PurEs. A contact between the AIR phosphate and a differentially conserved residue (TdPurE Lys41) enforces different AIR conformations in each PurE class. As a consequence, the TdPurE·AIR complex contains a portal that appears to allow the CO(2) substrate to enter the active site. In the human pathogen T. denticola, purine biosynthesis should depend on available CO(2) levels. Because spirochetes lack carbonic anhydrase, the corresponding reduction in bicarbonate demand may confer a selective advantage.

Novel pentadecenyl tetrazole enhances susceptibility of methicillin-resistant Staphylococcus aureus biofilms to gentamicin.

One method that bacteria employ to reduce their susceptibility to antibiotics is the formation of biofilms. We developed a robust 6-well plate biofilm assay to evaluate early-stage discovery compounds against methicillin-resistant Staphylococcus aureus (MRSA). Tissue culture-treated 6-well plates were selected for this assay because they facilitate the adherence of MRSA and enable accurate determination of the number of CFU in each well. The MRSA biofilms formed in this assay exhibit increased tolerances to clinically used antibiotics. Using this biofilm assay, we identified a novel potentiator of gentamicin against MRSA biofilms. The combination of gentamicin and pentadecenyl tetrazole is superior to clinically used MRSA antibiotics against these MRSA biofilms. This novel combination also exhibits synergistic effects on MRSA planktonic cells. This plant-derived compound reveals promise for its effectiveness and warrants further lead optimization as an antibiotic and aminoglycoside potentiator.

Optimization and qualification of an assay that demonstrates that a FimH vaccine induces functional antibody responses in women with histories of urinary tract infections.

Recurrent urinary tract infections (rUTI) are a serious disease associated with morbidities and mortality. Resistance to the standard of care antibiotics is now widespread because of the continued use of antibiotics among people who suffer from rUTI. We are therefore developing a vaccine to prevent recurrences among patients with rUTI. The antigen of the vaccine is FimH, a bacterial adhesin protein, and the vaccine is adjuvanted with a TLR-4 agonist. In a Phase 1 clinical study evaluating the vaccine, immunized individuals produced FimH-binding antibodies. Here we describe the optimization, qualification, and use of an assay to assess the functionality of these anti-FimH antibodies. The suitability of the assay for its intended purpose was demonstrated by selectivity, specificity, sensitivity, and intra-assay and inter-assay precision. The acceptance criteria were achieved for all parameters including intra-assay precision with ≤10% relative standard deviations and inter-assay precision with ≤25% relative standard deviations. The results presented herein suggest this functional assay will be important for supporting the vaccine's efficacy in future human studies. Furthermore and of great significance, these results prove that vaccine-induced functional antibodies can be elicited in rUTI patients against an essential virulence factor, FimH.

Safety and immunogenicity of an adjuvanted Escherichia coli adhesin vaccine in healthy women with and without histories of recurrent urinary tract infections: results from a first-in-human phase 1 study.

Antibiotic resistance among gram-negative bacteria continues to rise globally at an alarming rate. New vaccines that prevent bacterial infections and reduce antibiotic use could provide a potential solution to these problems. This study focused on development of an investigational vaccine to prevent recurrent urinary traction infections (UTI) caused by gram-negative bacteria that use type 1 pili to adhere to, invade, and colonize human bladders. The vaccine antigen is FimH, an adhesin protein on the tip of type 1 pili with a lectin binding domain that enables attachment to glycoproteins on mammalian bladders. This was a phase 1, open-label, dose escalation study evaluating the vaccine in 67 healthy women with and without histories of recurrent UTI. The objectives of the study were to evaluate the safety, tolerability, and immunogenicity of different dosages of the antigen and adjuvant of the vaccine. All dosages were well-tolerated and a low incidence of systemic reactions occurred. No serious adverse events related to the vaccine were reported. The vaccine induced both binding and functional antibodies. The women with histories of recurrent UTI demonstrated greater than 150-fold increases in antibodies against the N-terminal region of FimH. Based on the results of this phase 1 study, this vaccine is proceeding to a double-blind, randomized, placebo-controlled phase 2 study. If this vaccine is successful in future studies, it could potentially prevent millions of recurrent UTI globally and reduce the development of antibiotic resistance.

Antibiotic indole sesquiterpene alkaloid from Greenwayodendron suaveolens with a new natural product framework.

High-throughput natural products chemistry methods have led to the isolation of three new (1-3) and two known indole sesquiterpene alkaloids (4, 5) from Greenwayodendron suaveolens. Their structures were determined using CapNMR and MS. Pentacyclindole (1) was determined to possess a new natural product framework. Pentacyclindole (1) and polyalthenol (4) showed activity against clinical isolates of Staphylococcus aureus with polyalthenol (4) demonstrating a MIC(90) of 4 microg/mL.

Antibiofilm phenylethanoid glycosides from Penstemon centranthifolius.

Bioassay-guided fractionation of the antibacterial ethyl acetate-ethanol (50 : 50) extract obtained from the aerial parts of Penstemon centranthifolius led to the isolation of six phenylethanoid glycosides (1-6) and eleven iridoid glycosides (7-17). Their structures were determined on the basis of spectroscopic analysis and comparison with the literature. Among them, two phenylethanoid glycosides, 4'''-O-acetylverbascoside (1) and verbascoside (2), were found to show significant inhibition of the formation of bacterial biofilms by Escherichia coli UTI89. Compound 1 showed 77% biofilm inhibition at 2.5 microg/mL, and compound 2 showed 60% inhibition at 5 microg/mL.

Dammarane-type triterpene glycosides from Oncoba manii active against methicillin-resistant Staphylococcus aureus.

Drug-resistant bacteria are becoming more prevalent both in the community and in hospitals. In a search for new antibiotic leads, we used a high-throughput natural products chemistry approach to isolate one new (1) and two known (2, 3) dammarane-type triterpenes with mass-limited material from the African plant Oncoba manii. The new compound was determined by spectroscopic methods to be 1beta,2alpha,3beta,20(R)-tetrahydroxydammar-24-ene 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranoside. Compounds 1 and 2 inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA).

Bifidenone: Structure-Activity Relationship and Advanced Preclinical Candidate.

Bifidenone is a novel natural tubulin polymerization inhibitor that exhibits antiproliferative activity against a range of human cancer cell lines, making it an attractive candidate for development. A synthetic route was previously developed to alleviate supply constraints arising from its isolation in microgram quantities from a Gabonese tree. Using that previously published route, we present here 42 analogues that were synthesized to examine the structure-activity relationship of bifidenone derivatives. In addition to in vitro cytotoxicity data, data from murine xenograft and pharmacokinetic studies were used to evaluate the analogues. Compounds 45b and 46b were found to demonstrate promising efficacy in murine xenograft experiments, and 46b had significantly more potent in vitro antiproliferative activity against taxane-resistant cell lines compared to that of paclitaxel.