Synthesis of Monofluorinated 7-Hydroxycoumarin-3-Carboxamides as Cell-Permeable Fluorescent Molecular Probes.

To facilitate studies of engagement of protein targets by small molecules in living cells, we synthesized fluorinated derivatives of the fluorophore 7-hydroxycoumarin-3-carboxylic acid (7OHCCA). Compared to the related difluorinated coumarin Pacific Blue (PB), amide derivatives of 6-fluoro-7-hydroxycoumarin-3-carboxylic acid (6FC) exhibited substantially brighter fluorescence. When linked to the anticancer drug paclitaxel (Taxol) via gamma-aminobutyric acid (GABA), the acidity of the phenol of these coumarins profoundly affected cellular efflux and binding to microtubules in living cells. In contrast to the known fluorescent taxoid PB-GABA-Taxol, the less acidic 6FC-GABA-Taxol was more cell-permeable due to a lower susceptibility to active efflux. In living cells, this facilitated the imaging of microtubules by confocal microscopy and enabled quantification of binding to microtubules by flow cytometry without added efflux inhibitors. The photophysical, chemical, and biological properties of 6FC derivatives make these compounds particularly attractive for the construction of fluorescent molecular probes suitable for quantitative analysis of intracellular small molecule-protein interactions.

Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells.

The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (Kd) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular Kd values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular Ki values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular Ki values matched known biochemical Ki values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

BTK inhibition potentiates anti-PD-L1 treatment in murine melanoma: potential role for MDSC modulation in immunotherapy.

Myeloid-derived suppressor cells (MDSC) have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species and the production of inhibitory cytokines. Our group has shown that signaling through Bruton's tyrosine kinase (BTK) is important for MDSC function. Ibrutinib is an orally administered targeted agent that inhibits BTK activation and is currently used for the treatment of B cell malignancies. Using a syngeneic murine model of melanoma, the effect of BTK inhibition with ibrutinib on the therapeutic response to systemic PD-L1 blockade was studied. BTK was expressed by murine MDSC and their activation was inhibited by ibrutinib. Ibrutinib was not directly cytotoxic to cancer cells in vitro, but it inhibited BTK activation in MDSC and reduced expression of inducible nitric oxide synthase (NOS2) and production of nitric oxide. Ibrutinib treatments decreased the levels of circulating MDSC in vivo and increased the therapeutic efficacy of anti-PD-L1 antibody treatment. Gene expression profiling showed that ibrutinib decreased Cybb (NOX2) signaling, and increased IL-17 signaling (upregulating downstream targets Mmp9, Ptgs2, and S100a8). These results suggest that further exploration of MDSC inhibition could enhance the immunotherapy of advanced melanoma.PrécisInhibition of Bruton's tyrosine kinase, a key enzyme in myeloid cellular function, improves therapeutic response to an anti-PD-L1 antibody in an otherwise fairly resistant murine melanoma model.

Exploiting the fibroblast growth factor receptor-1 vulnerability to therapeutically restrict the MYC-EZH2-CDKN1C axis-driven proliferation in Mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a lethal hematological malignancy with a median survival of 4 years. Its lethality is mainly attributed to a limited understanding of clinical tumor progression and resistance to current therapeutic regimes. Intrinsic, prolonged drug treatment and tumor-microenvironment (TME) facilitated factors impart pro-tumorigenic and drug-insensitivity properties to MCL cells. Hence, elucidating neoteric pharmacotherapeutic molecular targets involved in MCL progression utilizing a global "unified" analysis for improved disease prevention is an earnest need. Using integrated transcriptomic analyses in MCL patients, we identified a Fibroblast Growth Factor Receptor-1 (FGFR1), and analyses of MCL patient samples showed that high FGFR1 expression was associated with shorter overall survival in MCL patient cohorts. Functional studies using pharmacological intervention and loss of function identify a novel MYC-EZH2-CDKN1C axis-driven proliferation in MCL. Further, pharmacological targeting with erdafitinib, a selective small molecule targeting FGFRs, induced cell-cycle arrest and cell death in-vitro, inhibited tumor progression, and improved overall survival in-vivo. We performed extensive pre-clinical assessments in multiple in-vivo model systems to confirm the therapeutic potential of erdafitinib in MCL and demonstrated FGFR1 as a viable therapeutic target in MCL.

Fluorescent Detection of Peroxynitrite Produced by Myeloid-Derived Suppressor Cells in Cancer and Inhibition by Dasatinib.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that expand dramatically in many cancer patients. This expansion contributes to immunosuppression in cancer and reduces the efficacy of immune-based cancer therapies. One mechanism of immunosuppression mediated by MDSCs involves production of the reactive nitrogen species peroxynitrite (PNT), where this strong oxidant inactivates immune effector cells through destructive nitration of tyrosine residues in immune signal transduction pathways. As an alternative to analysis of nitrotyrosines indirectly generated by PNT, we used an endoplasmic reticulum (ER)-targeted fluorescent sensor termed PS3 that allows direct detection of PNT produced by MDSCs. When the MDSC-like cell line MSC2 and primary MDSCs from mice and humans were treated with PS3 and antibody-opsonized TentaGel microspheres, phagocytosis of these beads led to production of PNT and generation of a highly fluorescent product. Using this method, we show that splenocytes from a EMT6 mouse model of cancer, but not normal control mice, produce high levels of PNT due to elevated numbers of granulocytic (PMN) MDSCs. Similarly, peripheral blood mononuclear cells (PBMCs) isolated from blood of human melanoma patients produced substantially higher levels of PNT than healthy human volunteers, coincident with higher peripheral MDSC levels. The kinase inhibitor dasatinib was found to potently block the production of PNT both by inhibiting phagocytosis in vitro and by reducing the number of granulocytic MDSCs in mice in vivo, providing a chemical tool to modulate the production of this reactive nitrogen species (RNS) in the tumor microenvironment.

Photoluminescent Properties and Mechanism of Novel Cyanine-Borondifluoride Curcuminoid Hybrids as Red-to-Near Infrared and Endoplasmic Reticulum-Targeting Dyes.

Synthesis-oriented design led us to the discovery of a series of novel cyanine-borondifluoride curcuminoid hybrids called Nanchang Red (NCR) dyes that overcome the intrinsic low synthetic yields of symmetrical cyanine-difluoroboronate (BF2 )-hybridized NIR dyes. The hybridization endows NCR dyes with high molar extinction coefficients, efficient red-to-NIR emission, and enlarged Stokes shifts. Quantum chemical calculations revealed that the asymmetrical layout of the three key electron-withdrawing and electron-donating fragments results in a special pattern of partial charge separation and inconsistent degrees of charge delocalization on their π-conjugated backbones. While the nature of the hemicyanine fragment exerts significant influence on the excitation modes of NCR dyes, the borondifluoride hemicurcuminoid fragment is the major contributor to the enlarged Stokes shifts. Cell imaging experiments illustrated that a subtle change in the N-heterocycle of the hemicyanine fragment has a remarkable effect on the subcellular localization of NCR dyes. Unlike other previously reported cyanine-BF2 hybridized dyes, which mainly target mitochondria, the benzothiazole and indole-based NCR dyes accumulate in both the endoplasmic reticulum (ER) and lipid droplets of HeLa cells, whereas the benzoxazole and quinoline-based NCR dyes stain the ER specifically.

Quantification of Engagement of Microtubules by Small Molecules in Living Cells by Flow Cytometry.

Drugs such as paclitaxel (Taxol) that bind microtubules are widely used for the treatment of cancer. Measurements of the affinity and selectivity of these compounds for their targets are largely based on studies of purified proteins, and only a few quantitative methods for the analysis of interactions of small molecules with microtubules in living cells have been reported. We describe here a novel method for rapidly quantifying the affinities of compounds that bind polymerized tubulin in living HeLa cells. This method uses the fluorescent molecular probe Pacific Blue-GABA-Taxol in conjunction with verapamil to block cellular efflux. Under physiologically relevant conditions of 37 °C, this combination allowed quantification of equilibrium saturation binding of this probe to cellular microtubules (K d = 1.7 µM) using flow cytometry. Competitive binding of the microtubule stabilizers paclitaxel (cellular K i = 22 nM), docetaxel (cellular K i = 16 nM), cabazitaxel (cellular K i = 6 nM), and ixabepilone (cellular K i = 10 nM) revealed intracellular affinities for microtubules that closely matched previously reported biochemical affinities. By including a cooperativity factor (α) for curve fitting of allosteric modulators, this probe also allowed quantification of binding (K b) of the microtubule destabilizers colchicine (K b = 80 nM, α = 0.08), vinblastine (K b = 7 nM, α = 0.18), and maytansine (K b = 3 nM, α = 0.21). Screening of this assay against 1008 NCI diversity compounds identified NSC 93427 as a novel microtubule destabilizer (K b = 485 nM, α = 0.02), illustrating the potential of this approach for drug discovery.

Synthesis of a fluorinated pyronin that enables blue light to rapidly depolarize mitochondria.

Fluorinated analogues of the fluorophore pyronin B were synthesized as a new class of amine-reactive drug-like small molecules. In water, 2,7-difluoropyronin B was found to reversibly react with primary amines to form covalent adducts. When this fluorinated analogue is added to proteins, these adducts undergo additional oxidation to yield fluorescent 9-aminopyronins. Irradiation with visible blue light enhances this oxidation step, providing a photochemical method to modify the biological properties of reactive amines. In living HeLa cells, 2,7-difluoropyronin B becomes localized in mitochondria, where it is partially transformed into fluorescent aminopyronins, as detected by spectral profiling confocal microscopy. Further excitation of these cells with the blue laser of a confocal microscope can depolarize mitochondria within seconds. This biological activity was only observed with 2,7-difluoropyronin B and was not detected with analogues such as pyronin B or 9-methyl-2,7-difluoropyronin B. This irradiation with blue light enhances the cellular production of reactive oxygen species (ROS), suggesting that increased ROS in mitochondria promotes the formation of aminopyronins that inactivate biomolecules critical for maintenance of mitochondrial membrane potential. The unique reactivity of 2,7-difluoropyronin B offers a novel tool for photochemical control of mitochondrial biology.

Pacific Blue-Taxoids as Fluorescent Molecular Probes of Microtubules.

Taxoids such as paclitaxel (Taxol) are an important class of anticancer drugs that bind ß-tubulin and stabilize cellular microtubules. To provide new chemical tools for studies of microtubules, we synthesized derivatives of paclitaxel modified at the 7-position with the small coumarin-derived fluorophore Pacific Blue (PB). Three of these Pacific Blue-Taxoids termed PB-Gly-Taxol, PB-ß-Ala-Taxol, and PB-GABA-Taxol bind purified crosslinked microtubules with affinities of 34-265 nM, where the affinity can be tuned based on the length of an amino acid linker. When added to living cells in the presence of verapamil or probenecid as inhibitors of efflux, these compounds allow visualization of the microtubule network by confocal microscopy. We describe methods for the synthesis of these probes, determination of their affinities for crosslinked tubulin, and imaging of microtubules in living HeLa cells. We further describe their uptake by Caco-2 cells and two transporter-deficient Caco-2 knockout cell lines in the absence and presence of efflux inhibitors by flow cytometry. These studies revealed that p-glycoprotein (MDR1) and multidrug-resistance protein 2 (MRP2) are major mediators of efflux of these molecular probes. These compounds provide useful tools for studies of microtubules and cellular efflux transporters in living cells.

Carnosine Protects Macrophages against the Toxicity of Aß1-42 Oligomers by Decreasing Oxidative Stress.

Carnosine (ß-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide has well-known antioxidant, anti-inflammatory, and anti-aggregation activities, and it may be useful for treatment of neurodegenerative disorders such as Alzheimer's disease (AD). In this disease, peripheral infiltrating macrophages play a substantial role in the clearance of amyloid beta (Aß) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aß have been reported. The effects of carnosine on macrophages and oxidative stress associated with AD are consequently of substantial interest for drug discovery in this field. In the present work, a model of stress induced by Aß1-42 oligomers was investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR. These assays were used to assess the ability of carnosine to protect macrophage cells, modulate oxidative stress, and profile the expression of genes related to inflammation and pro- and antioxidant systems. We found that pre-treatment of RAW 264.7 macrophages with carnosine counteracted cell death and apoptosis induced by Aß1-42 oligomers by decreasing oxidative stress as measured by levels of intracellular nitric oxide (NO)/reactive oxygen species (ROS) and production of peroxynitrite. This protective activity of carnosine was not mediated by modulation of the canonical inflammatory pathway but instead can be explained by the well-known antioxidant and free-radical scavenging activities of carnosine, enhanced macrophage phagocytic activity, and the rescue of fractalkine receptor CX3CR1. These new findings obtained with macrophages challenged with Aß1-42 oligomers, along with the well-known multimodal mechanism of action of carnosine in vitro and in vivo, substantiate the therapeutic potential of this dipeptide in the context of AD pathology.

Fluorescent detection of peroxynitrite during antibody-dependent cellular phagocytosis.

Peroxynitrite (PNT) is a highly reactive oxidant that plays a key role in the destruction of foreign pathogens by specific phagocytic immune cells such as macrophages. However, when its production is dysregulated, this oxidant can contribute to cardiovascular disease, neurological diseases, and cancer. To facilitate the detection of PNT in living cells, we designed and synthesized a fluorescent sensor termed PS3 that accumulates in membranes of the endoplasmic reticulum (ER). This subcellular targeting enhances the proximity of PS3 to the phagosome of macrophages where PNT is generated. When PS3-treated macrophages are stimulated with 10 µm opsonized tentagel microspheres, antibody-dependent cellular phagocytosis (ADCP) of these particles results in production of endogenous PNT, oxidative cleavage of the fluorescence-quenching phenolic side chain of PS3, and increased fluorescence that can be detected by confocal laser scanning microscopy, flow cytometry, and other assays. We describe methods for the synthesis of PS3 and evaluation of its photophysical properties, selectivity, and reactivity. We further report differential production of PNT during ADCP by the phagocytic cell lines RAW 264.7, J774A.1, and THP-1, as detected by confocal microscopy and changes in fluorescence intensity on 96-well plates. This approach may be useful for identification of modulators of PNT and related studies of ADCP.

Visible photorelease of liquid biopsy markers following microfluidic affinity-enrichment.

We detail a heterobifunctional, 7-aminocoumarin photocleavable (PC) linker with unique properties to covalently attach Abs to surfaces and subsequently release them with visible light (400-450 nm). The PC linker allowed rapid (2 min) and efficient (>90%) release of CTCs and EVs without damaging their molecular cargo.

The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen.

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previously, we developed a novel fluorescent dye (KR35) that accumulates in the intestine of C. elegans and reports a dynamic wave in intestinal pH associated with the defecation motor program. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1, abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that upon infection by pathogens, the intestinal pH becomes neutral in a wild type, but less so in pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby PBO-1 functions during infection to facilitate pH changes in the intestine that are protective to the host.

Synthetic Rhamnose Glycopolymer Cell-Surface Receptor for Endogenous Antibody Recruitment.

Synthetic materials capable of engineering the immune system are of great relevance in the fight against cancer to replace or complement the current monoclonal antibody and cell therapy-based immunotherapeutics. Here, we report on antibody recruiting glycopolymers (ARGPs). ARGPs consist of polymeric copies of a rhamnose motif, which can bind endogenous antirhamnose antibodies present in human serum. As a proof-of-concept, we have designed ARGPs with a lipophilic end group that efficiently inserts into cell-surface membranes. We validate the specificity of rhamnose to attract antibodies from human serum to the target cell surface and demonstrate that ARGPs outperform an analogous small-molecule compound containing only one single rhamnose motif. The ARGP concept opens new avenues for the design of potent immunotherapeutics that mark target cells for destruction by the immune system through antibody-mediated effector functions.

Antibody-Drug Conjugate that Exhibits Synergistic Cytotoxicity with an Endosome-Disruptive Peptide.

Antibody-drug conjugates are an important class of cancer therapeutics. These agents generally bind a specific cell surface receptor, undergo receptor-mediated endocytosis, and enter the endosomal-lysosomal system, where the environment in these organelles facilitates the release of a membrane-permeable cytotoxin. By using a membrane-impermeable cytotoxin, we describe here a method that allows the cytotoxicity of an antibody conjugate to be triggered by co-administration with an endosome-disruptive peptide that exhibits low toxicity. This approach was validated by conjugation of an anionic derivative of the tubulin-binding cytotoxin colchinol methyl ether to lysine residues of the HER2-targeting antibody trastuzumab (Herceptin) via a disulfide. When this antibody binds HER2 on SKBR3 breast cancer cells and undergoes endocytosis, the membrane-impermeable cytotoxin is released, but it becomes trapped in endosomes, resulting in relatively low cytotoxicity (IC50 > 1 µM). However, co-administration with an essentially nontoxic (IC50 > 10 µM) cholesterol-linked endosome-disruptive peptide promotes the release of this small molecule into the cytoplasm, conferring subnanomolar cytotoxic potency (IC50 = 0.11 ± 0.07 nM). Studies of a structurally related fluorophore conjugate revealed that the endosome-disruptive peptide does not substantially enhance cleavage of the disulfide (t 1/2 = 8 ± 2 h) within endosomes, suggesting that the mechanism of endosomal escape involves the efflux of some small molecules without facilitating substantial influx of reduced glutathione.

Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.

Targeting Fluorescent Sensors to Endoplasmic Reticulum Membranes Enables Detection of Peroxynitrite During Cellular Phagocytosis.

Peroxynitrite is a highly reactive oxidant derived from superoxide and nitric oxide. In normal vertebrate physiology, some phagocytes deploy this oxidant as a cytotoxin against foreign pathogens. To provide a new approach for detection of endogenous cellular peroxynitrite, we synthesized fluorescent sensors targeted to membranes of the endoplasmic reticulum (ER). The very high surface area of these membranes, approximately 30 times greater than the cellular plasma membrane, was envisioned as a vast intracellular platform for the display of sensors to transient reactive species. By linking an ER-targeted profluorophore to reactive phenols, sensors were designed to be cleaved by peroxynitrite and release a highly fluorescent ER-associated rhodol. Studies of kinetics in aqueous buffer revealed a linear free energy relationship where electron-donating substituents accelerate this reaction. However, in living cells, the efficiency of detection of endogenous cellular peroxynitrite was directly proportional to association with ER membranes. By incorporating a 2,6-dimethylphenol to accelerate the reaction and enhance this subcellular targeting, endogenous peroxynitrite in living RAW 264.7 macrophage cells could be readily detected after addition of antibody-opsonized tentagel microspheres, without additional stimulation, a process undetectable with other known fluorescent sensors. This approach provides uniquely sensitive tools for studies of transient reactive species in living mammalian cells.

Malleilactone Is a Burkholderia pseudomallei Virulence Factor Regulated by Antibiotics and Quorum Sensing.

Burkholderia pseudomallei, the causative agent of melioidosis, encodes almost a dozen predicted polyketide (PK) biosynthetic gene clusters. Many of these are regulated by LuxR-I-type acyl-homoserine (AHL) quorum-sensing systems. One of the PK gene clusters, the mal gene cluster, is conserved in the close relative Burkholderia thailandensis The B. thailandensis mal genes code for the cytotoxin malleilactone and are regulated by a genetically linked LuxR-type transcription factor, MalR. Although AHLs typically interact with LuxR-type proteins to modulate gene transcription, the B. thailandensis MalR does not appear to be an AHL receptor. Here, we characterize the mal genes and MalR in B. pseudomallei We use chemical analyses to demonstrate that the B. pseudomallei mal genes code for malleilactone. Our results show that MalR and the mal genes contribute to the ability of B. pseudomallei to kill Caenorhabditis elegans In B. thailandensis, antibiotics like trimethoprim can activate MalR by driving transcription of the mal genes, and we demonstrate that some of the same antibiotics induce expression of B. pseudomallei malR We also demonstrate that B. pseudomallei MalR does not respond directly to AHLs. Our results suggest that MalR is indirectly repressed by AHLs, possibly through a repressor, ScmR. We further show that malleilactone is a B. pseudomallei virulence factor and provide the foundation for understanding how malleilactone contributes to the pathology of melioidosis infections.IMPORTANCE Many bacterially produced polyketides are cytotoxic to mammalian cells and are potentially important contributors to pathogenesis during infection. We are interested in the polyketide gene clusters present in Burkholderia pseudomallei, which causes the often-fatal human disease melioidosis. Using knowledge gained by studies in the close relative Burkholderia thailandensis, we show that one of the B. pseudomallei polyketide biosynthetic clusters produces a cytotoxic polyketide, malleilactone. Malleilactone contributes to B. pseudomallei virulence in a Caenorhabditis elegans infection model and is regulated by an orphan LuxR family quorum-sensing transcription factor, MalR. Our studies demonstrate that malleilactone biosynthesis or MalR could be new targets for developing therapeutics to treat melioidosis.

Synthesis of a Fluorescent Analogue of Paclitaxel That Selectively Binds Microtubules and Sensitively Detects Efflux by P-Glycoprotein.

The anticancer drug paclitaxel (Taxol) exhibits paradoxical and poorly understood effects against slow-growing tumors. To investigate its biological activity, fluorophores such as Oregon Green have been linked to this drug. However, this modification increases its polarity by approximately 1000-fold and reduces the toxicity of Taxol towards cancer cell lines by over 200-fold. To construct more drug-like fluorescent probes suitable for imaging by confocal microscopy and analysis by flow cytometry, we synthesized derivatives of Taxol linked to the drug-like fluorophore Pacific Blue (PB). We found that PB-Gly-Taxol bound the target protein ß-tubulin with both high affinity in vitro and high specificity in living cells, exhibited substantial cytotoxicity towards HeLa cells, and was a highly sensitive substrate of the multidrug resistance transporter P-glycoprotein (P-gp).