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.

Exploring chemical diversity via a modular reaction pairing strategy.

The efficient synthesis of an 80-member library of unique benzoxathiazocine 1,1-dioxides by a microwave-assisted, intermolecular nucleophilic aromatic substitution (S(N)Ar) diversification pathway is reported. Eight benzofused sultam cores were generated by means of a sulfonylation/S(N)Ar/Mitsunobu reaction pairing protocol, and subsequently diversified by intermolecular S(N)Ar with ten chiral, non-racemic amine/amino alcohol building blocks. Computational analyses were employed to explore and evaluate the chemical diversity of the library.

Diverse methyl sulfone-containing benzo[b]thiophene library via iodocyclization and palladium-catalyzed coupling.

Parallel solution-phase methods for the synthesis of a 72-membered benzo[b]thiophene library are reported. Medicinally interesting, drug-like, methyl sulfone-substituted benzo[b]thiophenes have been prepared by the palladium-catalyzed substitution of 3-iodobenzo[b]thiophenes by Suzuki-Miyaura, Sonogashira, Heck, carboalkoxylation, and aminocarbonylation chemistry. The key intermediates for library generation, methyl sulfone-containing 3-iodobenzo[b]thiophenes, are readily prepared by iodocyclization and oxidation methodologies from readily available alkynes.

S(N)Ar-based, facile synthesis of a library of benzothiaoxazepine-1,1'-dioxides.

The construction of a library of benzothiaoxazepine-1,1'-dioxides utilizing a one-pot, S(N)Ar diversification-ODCT(50) scavenging protocol is reported. This protocol combines microwave irradiation to facilitate the reaction, in conjunction with a soluble ROMP-derived scavenger (ODCT) to afford the desired products in good overall purity. Utilizing this protocol, a 78-member library was successfully synthesized and submitted for biological evaluation.

Solution-phase parallel synthesis of a multi-substituted benzo[b]thiophene library.

Generation of a library using parallel syntheses of multi-substituted benzo[b]thiophenes is described. The requisite 3-iodobenzo[b]thiophenes are readily prepared in excellent yields from various alkynes bearing electron-rich aromatic rings by electrophilic cyclization using I(2) in CH(2)Cl(2). The heteroaromatic carbon-iodine bonds allow further diversification by palladium-catalyzed Suzuki-Miyaura, Sonogashira, Heck, and carboalkoxylation chemistry to give multi-substituted benzo[b]thiophene derivatives.

Palladium- and copper-catalyzed solution phase synthesis of a diverse library of isoquinolines.

The solution-phase synthesis of a 111 member isoquinoline library is described. The isoquinoline scaffold has been accessed through the palladium- and copper-catalyzed cyclization of iminoalkynes and the palladium-catalyzed iminoannulation of internal alkynes, followed by diversification of hydroxyl functionality where it is present.

Highly substituted indole library synthesis by palladium-catalyzed coupling reactions in solution and on a solid support.

3-Iodoindoles have been synthesized by the iodocyclization of N,N-dialkyl-o-(1-alkynyl)anilines, obtained by the Pd/Cu catalyzed coupling of terminal acetylenes with N,N-dialkyl-o-iodoanilines. These 3-iodoindoles undergo palladium-catalyzed Sonogashira and Suzuki coupling reactions to yield 1,2,3-trisubstituted indoles. These reactions have been applied to parallel library synthesis utilizing commercially available terminal acetylenes and boronic acids. The aforementioned chemistry has also been carried out on a chlorinated Wang resin as a solid support, affording 1,2,3,5-tetrasubstituted indoles after cleavage from the support. A diverse 42-member library of highly substituted indoles has been synthesized.

Solution-phase synthesis of a diverse isocoumarin library.

The solution-phase synthesis of a 167-member library of isocoumarins is described. The key intermediates for library generation, 4-iodoisocoumarins, are easily prepared by iodocyclization of the corresponding 2-(1-alkynyl)arenecarboxylate esters. The 4-iodoisocoumarins undergo palladium-catalyzed Sonogashira, Suzuki-Miyura, and Heck reactions to yield a diverse set of isocoumarins. Alternatively, isocoumarins, bearing hydroxyl or bromine functionalities, have been prepared by ZnCl(2)- and Pd(PPh(3))(4)-mediated cyclization of the corresponding o-iodobenzoic acid and appropriate terminal alkynes. The resulting isocoumarins were further diversified by derivatization of the hydroxyl or bromine groups. A small set of isoquinolinones were also prepared from the corresponding isocoumarins.

One-pot, three-component, domino Heck-aza-Michael approach to libraries of functionalized 1,1-dioxido-1,2-benzisothiazoline-3-acetic acids.

A sequential three-component synthesis of functionalized benzisothiazoline-3-acetic acid 1,1-dioxides utilizing a domino Heck-aza-Michael pathway is reported. This one-pot procedure rapidly assembles functionalized benzylsulfonamides, which undergo a palladium-catalyzed, domino, Heck-aza-Michael transformation in an experimentally straightforward manner. This attractive protocol has been utilized to synthesize three combinatorial sublibraries (I-III) comprising a total of 95 compounds in high purities (> or =95% for 75 compounds), yield and quantities.

Synthesis of a quinolone library from ynones.

A library of 72 quinolones was synthesized from substituted anthranilic acids, using ynone intermediates. These masked ß-dicarbonyl synthons allowed cyclization under milder conditions than previously reported quinolone syntheses.

Explorations of stemona alkaloid-inspired analogues: skeletal modification and functional group diversification.

A tandem Diels-Alder/Schmidt reaction provided an efficient route for the exploration of unnatural Stemona alkaloid analogues. Thus, a series of tricyclic scaffolds were efficiently prepared and then elaborated into seven sets of functionalized analogues. These derivatives incorporated appended heterocycles, such as indoles and quinolines, or other diversity-incorporating moieties such as carbamates and amines. Both the scaffold-generation sequence and the diversification steps could be manipulated to provide regio- and diastereochemically pure products.

Solution-phase parallel synthesis of hexahydro-1H-isoindolone libraries via tactical combination of Cu-catalyzed three-component coupling and Diels-Alder reactions.

Parallel solution-phase synthesis of combinatorial libraries of hexahydro-1 H-isoindolones exploiting a novel "tactical combination" of Cu-catalyzed three-component coupling and Diels-Alder reactions was accomplished. Three distinct libraries consisting of 24 members (library I), 60 members (library II), and 32 members (library III) were constructed. Variation of three substituents on the isoindolone scaffold in library I was exclusively achieved by the choice of the building blocks. In the syntheses of libraries II and III, sublibraries of isoindolone scaffolds were prepared initially in a one-pot/two-step process and were further diversified via Pd-catalyzed Suzuki cross-coupling reaction with boronic acids at two different diversification points. The Lipinski profiles and calculated ADME properties of the compounds are also reported.

Efficient synthesis of gamma-lactams by a tandem reductive amination/lactamization sequence.

A three-component method for the synthesis of highly substituted gamma-lactams from readily available maleimides, aldehydes, and amines is described. A new reductive amination/intramolecular lactamization sequence provides a straightforward route to the lactam products in a single manipulation. The general utility of this method is demonstrated by the parallel synthesis of a gamma-lactam library.

Parallel synthesis of a multi-substituted benzo[b]furan library.

The solution-phase parallel synthesis of a 121-member library of multi-substituted benzo[ b]furans is described. 2,3,5-Trisubstituted benzo[ b]furans have been prepared by the palladium-catalyzed substitution of 3-iodobenzofurans by Suzuki-Miyaura, carbonylative Suzuki, Sonogashira, Heck, and carboalkoxylation chemistry. The 3-iodobenzofurans are readily prepared in good to excellent yields by the palladium/copper-catalyzed cross-coupling of various o-iodoanisoles and terminal alkynes, followed by electrophilic cyclization with ICl.

Solution phase synthesis of a diverse library of highly substituted isoxazoles.

The iodocyclization of O-methyloximes of 2-alkyn-1-ones affords 4-iodoisoxazoles, which undergo various palladium-catalyzed reactions to yield 3,4,5-trisubstituted isoxazoles. The palladium-catalyzed processes have been adapted to parallel synthesis utilizing commercially available boronic acid, acetylene, styrene, and amine sublibraries. Accordingly, a diverse 51-member library of 3,4,5-trisubstituted isoxazoles has been generated.

Ionic immobilization, diversification, and release: application to the generation of a library of methionine aminopeptidase inhibitors.

Development of an ionic immobilization, diversification, and release method for the generation of methionine aminopeptidase inhibitors is reported. This method involves the immobilization of 5-bromofuran-2-carboxylic acid and 5-bromothiophene-2-carboxylic acid onto PS-BEMP, followed by Suzuki reaction on a resin-bound intermediate and subsequent release to provide products in moderate yields and excellent purities. Compound potencies were evaluated on the Co(II), Mn(II), Ni(II), and Fe(II) forms of Escherichia coli MetAP1. The furoic-acid analogs were found to be Mn(II) selective with IC 50 values in the low micromolar range. Qualitative SAR analysis, supplemented by molecular modeling studies, provides valuable information on structural elements responsible for potency and selectivity.

Quorum-sensing control of antibiotic resistance stabilizes cooperation in Chromobacterium violaceum.

Many Proteobacteria use quorum sensing to regulate production of public goods, such as antimicrobials and proteases, that are shared among members of a community. Public goods are vulnerable to exploitation by cheaters, such as quorum sensing-defective mutants. Quorum sensing- regulated private goods, goods that benefit only producing cells, can prevent the emergence of cheaters under certain growth conditions. Previously, we developed a laboratory co-culture model to investigate the importance of quorum-regulated antimicrobials during interspecies competition. In our model, Burkholderia thailandensis and Chromobacterium violaceum each use quorum sensing-controlled antimicrobials to inhibit the other species' growth. Here, we show that C. violaceum uses quorum sensing to increase resistance to bactobolin, a B. thailandensis antibiotic, by increasing transcription of a putative antibiotic efflux pump. We demonstrate conditions where C. violaceum quorum-defective cheaters emerge and show that in these conditions, bactobolin restrains cheaters. We also demonstrate that bactobolin restrains quorum-defective mutants in our co-culture model, and the increase in antimicrobial-producing cooperators drives the C. violaceum population to become more competitive. Our results describe a mechanism of cheater restraint involving quorum control of efflux pumps and demonstrate that interspecies competition can reinforce cooperative behaviors by placing constraints on quorum sensing-defective mutants.

The C-2 derivatives of salvinorin A, ethoxymethyl ether Sal B and ß-tetrahydropyran Sal B, have anti-cocaine properties with minimal side effects.

RATIONALE: Kappa-opioid receptor (KOPr) agonists have pre-clinical anti-cocaine and analgesic effects. However, side effects including sedation, dysphoria, aversion, anxiety and depression limit their therapeutic development. The unique structure of salvinorin A has been used to develop longer acting KOPr agonists. OBJECTIVES: We evaluate two novel C-2 analogues of salvinorin A, ethoxymethyl ether Sal B (EOM Sal B) and ß-tetrahydropyran Sal B (ß-THP Sal B) alongside U50,488 for their ability to modulate cocaine-induced behaviours and side effects, pre-clinically. METHODS: Anti-cocaine properties of EOM Sal B were evaluated using the reinstatement model of drug seeking in self-administering rats. EOM Sal B and ß-THP Sal B were evaluated for effects on cocaine-induced hyperactivity, spontaneous locomotor activity and sucrose self-administration. EOM Sal B and ß-THP Sal B were evaluated for aversive, anxiogenic and depressive-like effects using conditioned place aversion (CPA), elevated plus maze (EPM) and forced swim tests (FSTs), respectively. RESULTS: EOM Sal B (0.1, 0.3 mg/kg, intraperitoneally (i.p.)) dose dependently attenuated drug seeking, and EOM Sal B (0.1 mg/kg, i.p.) and ß-THP Sal B (1 mg/kg, i.p.) attenuated cocaine-induced hyperactivity. No effects on locomotor activity, open arm times (EPM) or swimming behaviours (FST) were seen with EOM (0.1 or 0.3 mg/kg, i.p.) or ß-THP Sal B (1 or 2 mg/kg, i.p.). However, ß-THP Sal B decreased time spent in the drug-paired chamber. CONCLUSION: EOM Sal B is more potent than Sal A and ß-THP Sal B in reducing drug-seeking behaviour with fewer side effects. EOM Sal B showed no effects on sucrose self-administration (0.1 mg/kg), locomotor, depressive-like, aversive-like or anxiolytic effects.

Addressing Structural Flexibility at the A-Ring on Salvinorin A: Discovery of a Potent Kappa-Opioid Agonist with Enhanced Metabolic Stability.

Previous structure-activity studies on the neoclerodane diterpenoid salvinorin A have demonstrated the importance of the acetoxy functionality on the A-ring in its activity as a κ-opioid receptor agonist. Few studies have focused on understanding the role of conformation in these interactions. Herein we describe the synthesis and evaluation of both flexible and conformationally restricted compounds derived from salvinorin A. One such compound, spirobutyrolactone 14, was synthesized in a single step from salvinorin B and had similar potency and selectivity to salvinorin A (EC50 = 0.6 ± 0.2 nM at κ; >10000 nM at µ and δ). Microsomal stability studies demonstrated that 14 was more metabolically resistant than salvinorin A. Evaluation of analgesic and anti-inflammatory properties revealed similar in vivo effects between 14 and salvinorin A. To our knowledge, this study represents the first example of bioisosteric replacement of an acetate group by a spirobutyrolactone to produce a metabolically resistant derivative.

N-Phenylbenzamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore.

Persistent opening of the mitochondrial permeability transition pore (PTP), an inner membrane channel, leads to mitochondrial dysfunction and renders the PTP a therapeutic target for a host of life-threatening diseases. Herein, we report our effort toward identifying small-molecule inhibitors of this target through structure-activity relationship optimization studies, which led to the identification of several potent analogues around the N-phenylbenzamide compound series identified by high-throughput screening. In particular, compound 4 (3-(benzyloxy)-5-chloro-N-(4-(piperidin-1-ylmethyl)phenyl)benzamide) displayed noteworthy inhibitory activity in the mitochondrial swelling assay (EC50 =280 nm), poor-to-very-good physicochemical as well as in vitro pharmacokinetic properties, and conferred very high calcium retention capacity to mitochondria. From the data, we believe compound 4 in this series represents a promising lead for the development of PTP inhibitors of pharmacological relevance.