Showing papers by "Jon Clardy published in 2016"

Diversity-oriented synthesis yields novel multistage antimalarial inhibitors

Abstract: Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Dynamic metabolic exchange governs a marine algal-bacterial interaction

Abstract: Microscopic algae that live in the ocean release countless tons of oxygen into the atmosphere each year. Widespread algae – known as coccolithophores – surround their little plant-like body with a mineral shell made of a material similar to chalk. These microscopic algae form seasonal blooms. Over several weeks in early summer, the algae grow to enormous numbers and cover hundreds of thousands of square kilometers in the ocean. These blooms become so vast that satellites can detect them. However, suddenly the blooms collapse; the algae die and their chalky shells sink to the bottom of the ocean where they have been accumulating for millions of years. More and more evidence suggests that these tiny algae interact with bacteria in various ways. However, so far, no one had documented a direct interaction between bacteria and a member of this key group of algae. Now, in a controlled laboratory environment, Segev et al. show that marine bacteria from the Roseobacter group physically attach onto a tiny coccolithophore alga called Emiliania huxleyi. While the bacteria are attached to their algal host, they enjoy a supply of nutrients that trickles from the algal cell. Unexpectedly, Segev et al. also discovered that the algae grow better in the presence of the bacteria. It turns out that the bacteria use a molecule that they obtain from their algal hosts to produce a small hormone-like molecule that in turn enhances the growth of the algae. However, after three weeks of growing together, the bacteria produce so much of the growth-enhancing molecule – which is harmful in higher concentrations – that they actually kill their algal host. These findings suggest that the bacteria first promote the alga’s growth to boost their supply of nutrients. But as algae grow older, the bacteria harvest the algae to enjoy a last pulse of nutrients and allow their offspring to swim away and attach to younger algae. The next challenge will be to link these laboratory observations to the actual microbial interactions in the ocean. It will also be important to explore whether other algae and bacteria interact in similar ways and if bacteria contribute to the sudden collapse of algal blooms by killing the algae.

Bacterial lipids activate, synergize, and inhibit a developmental switch in choanoflagellates

Abstract: In choanoflagellates, the closest living relatives of animals, multicellular rosette development is regulated by environmental bacteria. The simplicity of this evolutionarily relevant interaction provides an opportunity to identify the molecules and regulatory logic underpinning bacterial regulation of development. We find that the rosette-inducing bacterium Algoriphagus machipongonensis produces three structurally divergent classes of bioactive lipids that, together, activate, enhance, and inhibit rosette development in the choanoflagellate Salpingoeca rosetta. One class of molecules, the lysophosphatidylethanolamines (LPEs), elicits no response on its own but synergizes with activating sulfonolipid rosette-inducing factors (RIFs) to recapitulate the full bioactivity of live Algoriphagus. LPEs, although ubiquitous in bacteria and eukaryotes, have not previously been implicated in the regulation of a host-microbe interaction. This study reveals that multiple bacterially produced lipids converge to activate, enhance, and inhibit multicellular development in a choanoflagellate.

Homodimericin A: A Complex Hexacyclic Fungal Metabolite

Abstract: Microbes sense and respond to their environment with small molecules, and discovering these molecules and identifying their functions informs chemistry, biology, and medicine. As part of a study of molecular exchanges between termite-associated actinobacteria and pathogenic fungi, we uncovered a remarkable fungal metabolite, homodimericin A, which is strongly upregulated by the bacterial metabolite bafilomycin C1. Homodimericin A is a hexacyclic polyketide with a carbon backbone containing eight contiguous stereogenic carbons in a C20 hexacyclic core. Only half of its carbon atoms have an attached hydrogen, which presented a significant challenge for NMR-based structural analysis. In spite of its microbial production and rich stereochemistry, homodimericin A occurs naturally as a racemic mixture. A plausible nonenzymatic reaction cascade leading from two identical achiral monomers to homodimericin A is presented, and homodimericin A’s formation by this path, a six-electron oxidation, could be a response t...

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts

Abstract: The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin’s shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin’s biosynthesis including a subcluster of genes consistent with selvamicin’s 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.

Compounds that select against the tetracycline-resistance efflux pump.

Abstract: We developed a competition-based screening strategy to identify compounds that invert the selective advantage of antibiotic resistance. Using our assay, we screened over 19,000 compounds for the ability to select against the TetA tetracycline-resistance efflux pump in Escherichia coli and identified two hits, β-thujaplicin and disulfiram. Treating a tetracycline-resistant population with β-thujaplicin selects for loss of the resistance gene, enabling an effective second-phase treatment with doxycycline.

A common bacterial metabolite elicits prion-based bypass of glucose repression

Abstract: We communicate with each other using speech, writing and physical gestures. But how do bacteria, yeast and other single-celled microbes communicate? In 2014, researchers reported a new example of communication between bacteria and yeast in which the bacteria send a chemical message that has a very long-lasting effect on how the yeast grow in certain environments. This in turn also affected the ability of the bacteria to survive in these environments. The identity of the chemical message produced by the bacteria, however, was not known. Garcia, Dietrich et al. – including one of the researchers from the previous study – used biochemical and genetic approaches to identify the chemical message. The experiments show that the message is a molecule called lactic acid, which is very common in nature and is produced by many bacteria. Garcia, Dietrich et al. found out how much lactic acid is needed to alter the growth of brewer’s yeast, and which genes in yeast are involved in responding to the message from the bacteria. Further experiments suggest that the ability of yeast and bacteria to communicate using lactic acid is likely to have existed for hundreds of millions of years. The next step following this work will be to identify other chemical messages used by microbes. The human body is packed with billions of bacterial cells, and in some cases yeast can also take up residence. A future challenge will be to find out if bacteria and yeast inside the human body are able to communicate with each other in ways that could affect our health.

Total chemical synthesis of lassomycin and lassomycin-amide

Abstract: Herein we report a practical synthetic route to the lasso peptide lassomycin (1) and C-terminal variant lassomycin-amide (2). The biological evaluation of peptides 1 and 2 against Mycobacterium tuberculosis revealed that neither had any activity against this bacterium. This lack of biological activity has led us to propose that naturally occurring lassomycin may actually exhibit a standard lasso peptide threaded conformation rather than the previously reported unthreaded structure.

Isolation and Synthesis of a Bacterially Produced Inhibitor of Rosette Development in Choanoflagellates

Abstract: The choanoflagellate Salpingoeca rosetta is a microbial marine eukaryote that can switch between unicellular and multicellular states. As one of the closest living relatives of animals, this organism has become a model for understanding how multicellularity evolved in the animal lineage. Previously our laboratories isolated and synthesized a bacterially produced sulfonolipid that induces S. rosetta to form multicellular “rosettes.” In this study, we report the identification of a bacterially produced inhibitor of rosettes (IOR-1) as well as the total synthesis of this molecule and all of its stereoisomers. Our results confirm the previously noted specificity and potency of rosette-modulating molecules, expand our understanding of the complex chemical ecology between choanoflagellates and rosette-inducing bacteria, and provide a synthetic probe template for conducting further mechanistic studies on the emergence of multicellularity.

Discovery of Dual Stage Malaria Inhibitors with New Targets

Abstract: Malaria remains a major global health problem, with more than half of the world population at risk of contracting the disease and nearly a million deaths each year. Here, we report the discovery of inhibitors that target multiple stages of malaria parasite growth. To identify these inhibitors, we took advantage of the Tres Cantos Antimalarial Compound Set (TCAMS) small-molecule library, which is comprised of diverse and potent chemical scaffolds with activities against the blood stage of the malaria parasite, and investigated their effects against the elusive liver stage of the malaria parasite using a forward chemical screen. From a screen of nearly 14,000 compounds, we identified and confirmed 103 compounds as dual-stage malaria inhibitors. Interestingly, these compounds show preferential inhibition of parasite growth in liver- versus blood-stage malaria parasite assays, highlighting the drug susceptibility of this parasite form. Mode-of-action studies were completed using genetically modified and drug-resistant Plasmodium parasite strains. While we identified some compound targets as classical antimalarial pathways, such as the mitochondrial electron transport chain through cytochrome bc1 complex inhibition or the folate biosynthesis pathway, most compounds induced parasite death through as yet unknown mechanisms of action. Importantly, the identification of new chemotypes with different modes of action in killing Plasmodium parasites represents a promising opportunity for probing essential and novel molecular processes that remain to be discovered. The chemical scaffolds identified with activity against drug-resistant Plasmodium parasites represent starting points for dual-stage antimalarial development to surmount the threat of malaria parasite drug resistance.

Natural Product Screening Reveals Naphthoquinone Complex I Bypass Factors.

Abstract: Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS). Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as “complex I bypass.” In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology.

Whole-Genome Sequence of Bacillus sp. SDLI1, Isolated from the Social Bee Scaptotrigona depilis

Abstract: ABSTRACT We announce the complete genome sequence of Bacillus sp. strain SDLI1, isolated from larval gut of the stingless bee Scaptotrigona depilis. The 4.13-Mb circular chromosome harbors biosynthetic gene clusters for the production of antimicrobial compounds.

Microbial Symbionts of Insects are the Focus of the First International Cooperative Biodiversity Group (ICBG) in Brazil

Abstract: The basic aim of the ICBG (International Cooperative Biodiversity Group) program is to benefit both the host country and the global scientific community by discovering and developing new solutions to human health problems based on previously unexplored genetic resources. The first ICBG in Brazil is jointly supported by the Fogarty International Center of the National Institutes of Health (FIC/NIH) in the USA and by the Sao Paulo Research Foundation (FAPESP) in Brazil. The ongoing ICBG, developed under the BIOTA-FAPESP Program, is based on the highly evolved fungus-growing ant multilateral symbiosis between three mutualists and one parasite. The project aligns chemical ecology and therapeutic application, increasing the chances of discovering new antifungal, antibacterial, anticancer and antiprotozoal hits and leads. In this article we describe the rationale of the ICBG and the legal requirements to develop this type of international collaborative project in Brazil.

Whole-Genome Sequence of Bacillus sp. SDLI1, Isolated from the Social Bee Scaptotrigona depilis.

Abstract: We announce the complete genome sequence of Bacillus sp. strain SDLI1, isolated from larval gut of the stingless bee Scaptotrigona depilis. The 4.13-Mb circular chromosome harbors biosynthetic gene clusters for the production of antimicrobial compounds.

Genome Sequence of Streptomyces sp. Strain RTd22, an Endophyte of the Mexican Sunflower.

Abstract: We report here the complete genome sequence of Streptomyces sp. strain RTd22, an endophytic actinobacterium that was isolated from the roots of the Mexican sunflower Tithonia diversifolia. The bacterium9s 11.1-Mb linear chromosome is predicted to encode a large number of unknown natural products.

Draft Genome Sequence of Shewanella sp. Strain P1-14-1, a Bacterial Inducer of Settlement and Morphogenesis in Larvae of the Marine Hydroid Hydractinia echinata

Abstract: The assembly and annotation of the draft genome sequence of Shewanella sp. strain P1-14-1 are reported here to investigate the genes responsible for interkingdom interactions, secondary metabolite production, and microbial electrogenesis.

Iridoid and phenylethanoid glycosides from the aerial part of Barleria lupulina

Abstract: A new iridoid glycoside, barlupulin C methyl ester (1), together with two known phenylethanoid glycosides (2 and 3) and three known simple phenolic glycosides (4-6) were isolated from the aerial parts of Barleria lupulina Lindl., Acanthaceae. The structure of the new compound (1) was elucidated through 1D and 2D NMR spectroscopic data, and HR-ESIMS. Interestingly, compound (1) has a formate group attached to the C-6 hydroxy group of the glucose unit. Compounds 2-6 were identified as poliumoside (2), decaffeoylacteoside (3), protocatechuic acid 4-O-β-glucoside (4), vanillic acid 4-O-β-glucoside (5), and leonuriside A (6) on the basis of NMR spectroscopic data analyses and comparison with those reported in the literature. Compounds 3-6 were isolated from B. lupulina for the first time.

Isolation and Synthesis of a Bacterially Produced Inhibitor of Rosette Development in Choanoflagellates.

Abstract: The choanoflagellate Salpingoeca rosetta is a microbial marine eukaryote that can switch between unicellular and multicellular states. As one of the closest living relatives of animals, this organism has become a model for understanding how multicellularity evolved in the animal lineage. Previously our laboratories isolated and synthesized a bacterially produced sulfonolipid that induces S. rosetta to form multicellular “rosettes.” In this study, we report the identification of a bacterially produced inhibitor of rosettes (IOR-1) as well as the total synthesis of this molecule and all of its stereoisomers. Our results confirm the previously noted specificity and potency of rosette-modulating molecules, expand our understanding of the complex chemical ecology between choanoflagellates and rosette-inducing bacteria, and provide a synthetic probe template for conducting further mechanistic studies on the emergence of multicellularity.

Stilbenoids from Hopea acuminata

Abstract: Two resveratrol oligomers, a dimer (1) and a tetramer (2), were isolated from the ethyl acetate extracts of the leaves of Hopea acuminata along with vaticanol B, vaticaphenol A, vateriaphenol B, hopeaphenol, e-viniferindiol, balanocarpol, and ampelopsin A. The structures of these compounds were established on the basis of spectroscopic data including two-dimentional nuclear magnetic resonance experiments. Compound 2, vaticanol B, vaticaphenol A, vateriaphenol B, hopeaphenol were found to inhibit protein splicing mediated by the Mycobacterium tuberculosis RecA intein in a nonspecific manner.

A New Diketopiperazine, Cyclo(D-trans-Hyp-L-Leu) from a Kenyan Bacterium Bacillus licheniformis LB 8CT:

Abstract: Bacterially-produced small molecules demonstrate a wide range of structural and functional diversity. A new diketopiperazine, cyclo(D-trans-Hyp-L-Leu) (1), and five other known diketopiperazines (2-6), were isolated and purified from the fermented broth of a Kenyan bacterium Bacillus licheniformis LB 8CT. The structure of 1 was elucidated by a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS, and the absolute configuration was determined by a combination of NOESY analysis and Marfey's method. The known compounds were identified as cyclo(D-cis-Hyp-L-Leu) (2), cyclo(D-cis-Hyp-L-Phe) (3), cyclo(D-Pro-L-Tyr) (4), cyclo-(D-Trp-L-Leu) (5), and cyclo(L-Tyr-Gly) (6) by comparison of their spectroscopic and physical data with reported values. Compounds 1-6 were tested for antifungal and antimicrobial properties.

Genome Sequence of Streptomyces sp. Strain RTd22, an Endophyte of the Mexican Sunflower

Abstract: We report here the complete genome sequence of Streptomyces sp. strain RTd22, an endophytic actinobacterium that was isolated from the roots of the Mexican sunflower Tithonia diversifolia. The bacterium9s 11.1-Mb linear chromosome is predicted to encode a large number of unknown natural products.

Diketopiperazines from Costa Rican Endolichenic Fungus Colpoma sp. CR1465A

Abstract: Three new diketopiperazines (1-3), cyclo(l-Pro-d-trans-Hyp) (1), cyclo(l-Pro-d-Glu) (2), and cyclo(d-Pro-d-Glu) (3) and five known diketopiperazines (4-8) were isolated from the endolichenic fungus Colpoma sp. CR1465A identified from the Costa Rican plant Henriettea tuberculosa (Melatomataceae). The structures of the new compounds 1-3 were elucidated using a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS, and their absolute configurations were determined by a combination of NOESY analysis and Marfey's method. Cyclo(l-Pro-d-allo-Thr) (4) was recently isolated from a South China Sea marine sponge Callyspongia sp., but its NMR spectroscopic data were not reported, and cyclo(l-Pro-l-Asp) (5) was previously reported but only as a synthetic product. The NMR data assignments of compounds 4 and 5 are reported for the first time. All of the isolated compounds were tested for antifungal and antimicrobial properties.

Bacteria regulate choanoflagellate development with lipid activators, inhibitors, and synergists

Abstract: In choanoflagellates, the closest living relatives of animals, multicellular 'rosette' development is regulated by environmental bacteria. The simplicity of this evolutionarily-relevant interaction provides an opportunity to identify the molecules and regulatory logic underpinning bacterial regulation of development. We find that the rosette-inducing bacterium Algoriphagus machipongonensis produces three structurally divergent classes of bioactive lipids that, together, activate, enhance, and inhibit rosette development in the choanoflagellate S. rosetta. One class of molecules, the lysophosphatidylethanolamines (LPEs), elicits no response on its own, but synergizes with activating sulfonolipid rosette inducing factors (RIFs) to recapitulate the full bioactivity of live Algoriphagus. LPEs, while ubiquitous in bacteria and eukaryotes, have not previously been implicated in the regulation of a host-microbe interaction. This study reveals that multiple bacterially-produced lipids converge to activate, enhance, and inhibit multicellular development in a choanoflagellate.