R06. Identification of the Cellular Pathways Targeted by Two Antifungal Natural Products Using RNA-Seq Analysis

Corresponding author (NCNPR): Ameeta Agarwal, [email protected]://egrove.olemiss.edu/pharm_annual_posters/1005/thumbnail.jp

Synthesis of Natural Acylphloroglucinol-Based Antifungal Compounds against <i>Cryptococcus</i> Species

Thirty-three natural-product-based acylphloroglucinol derivatives were synthesized to identify antifungal compounds against <i>Cryptococcus</i> spp. that cause the life-threatening disseminated cryptococcosis. In vitro antifungal testing showed that 17 compounds were active against <i>C. neoformans</i> ATCC 90113, <i>C. neoformans</i> H99, and <i>C. gattii</i> ATCC 32609, with minimum inhibitory concentrations (MICs) in the range 1.0–16.7 μg/mL. Analysis of the structure and antifungal activity of these compounds indicated that the 2,4-diacyl- and 2-acyl-4-alkylphloroglucinols were more active than <i>O</i>-alkyl-acylphloroglucinols. The most promising compound found was 2-methyl-1-(2,4,6-trihydroxy-3-(4-isopropylbenzyl)­phenyl)­propan-1-one (<b>11j</b>), which exhibited potent antifungal activity (MICs, 1.5–2.1 μg/mL) and low cytotoxicity against the mammalian Vero and LLC-PK1 cell lines (IC₅₀ values >50 μg/mL). This compound may serve as a template for further synthesis of new analogues with improved antifungal activity. The findings of the present work may contribute to future antifungal discovery toward pharmaceutical development of new treatments for cryptococcosis

Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol (Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acety1-3-butanoyl-chloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1-p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 mu g/mL, respectively.Identification of anti-microbial agents from metagenomic clone

Antibacterial Prenylated Acylphloroglucinols from <i>Psorothamnus fremontii</i>

Psorothatins A–C (<b>1</b>–<b>3</b>), three antibacterial prenylated acylphloroglucinol derivatives, were isolated from the native American plant <i>Psorothamnus fremontii</i>. They feature an unusual α,β-epoxyketone functionality and a β-hydroxy-α,β-unsaturated ketone structural moiety. The latter forms a pseudo-six-membered heterocyclic ring due to strong intramolecular hydrogen bonding, as indicated by the long-range proton–carbon correlations in the NMR experiments. Psorothatin C (<b>3</b>) was the most active compound against methicillin-resistant <i>Staphylococcus aureus</i> and vancomycin-resistant <i>Enterococcus faecium</i>, with IC₅₀ values in the range 1.4–8.8 μg/mL. The first total synthesis of <b>3</b> described herein permits future access to structural analogues with potentially improved antibacterial activities

Bacillusin A, an Antibacterial Macrodiolide from <i>Bacillus amyloliquefaciens</i> AP183

Bioassay-guided fractionation of the organic extracts of a <i>Bacillus amyloliquefaciens</i> strain (AP183) led to the discovery of a new macrocyclic polyene antibiotic, bacillusin A (<b>1</b>). Its structure was assigned by interpretation of NMR and MS spectroscopic data as a novel macrodiolide composed of dimeric 4-hydroxy-2-methoxy-6-alkenylbenzoic acid lactones with conjugated pentaene-hexahydroxy polyketide chains. Compound <b>1</b> showed potent antibacterial activities against methicillin-resistant <i>Staphylococcus aureus</i> and vancomycin-resistant <i>Enterococcus faecium</i> with minimum inhibitory concentrations in a range of 0.6 to 1.2 μg/mL. The biosynthetic significance of this unique class of antibiotic compounds is briefly discussed

LC-MS- and ¹H NMR Spectroscopy-Guided Identification of Antifungal Diterpenoids from <i>Sagittaria latifolia</i>

Antifungal screening of small-molecule natural product libraries showed that a column fraction (CF) derived from the plant extract of <i>Sagittaria latifolia</i> was active against the fungal pathogen <i>Cryptococcus neoformans</i>. Dereplication analysis by liquid chromatography–mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy (¹H NMR) indicated the presence of new compounds in this CF. Subsequent fractionation of the plant extract resulted in the identification of two new isopimaradiene-type diterpenoids, <b>1</b> and <b>2</b>. The structures of <b>1</b> and <b>2</b> were determined by chemical methods and spectroscopic analysis as isopimara-7,15-dien-19-ol 19-<i>O</i>-α-l-arabinofuranoside and isopimara-7,15-dien-19-ol 19-<i>O</i>-α-l-(5′-acetoxy)­arabinofuranoside, respectively. Compound <b>1</b> exhibited IC₅₀ values of 3.7 and 1.8 μg/mL, respectively, against <i>C. neoformans and C. gattii</i>. Its aglycone, isopimara-7,15-dien-19-ol (<b>3</b>), resulting from acid hydrolysis of <b>1</b>, was also active against the two fungal pathogens, with IC₅₀ values of 9.2 and 6.8 μg/mL, respectively. This study demonstrates that utilization of the combined LC-MS and ¹H NMR analytical tools is an improved chemical screening approach for hit prioritization in natural product drug discovery

Biological evaluation of phytoconstituents from <i>Polygonum hydropiper</i>

<p>Fourteen compounds including vanicoside B (<b>1</b>), vanicoside F (<b>2</b>), vanicoside E (<b>3</b>) and 5,6-dehydrokawain (<b>4</b>), aniba-dimer-A (<b>5</b>), 6,6′-((1<i>α</i>,2<i>α</i>,3<i>β</i>,4<i>β</i>)-2,4-diphenylcyclobutane-1,3-diyl)bis(4-methoxy-2<i>H</i>-pyran-2-one) (<b>6</b>), (+)-ketopinoresinol (<b>7</b>), isorhamnetin (<b>8</b>), 3,7-dihydroxy-5,6-dimethoxy-flavone (<b>9</b>), isalpinin (<b>10</b>), cardamomin (<b>11</b>), pinosylvin (<b>12</b>), 2-desoxy-4-<i>epi</i>-pulchellin (<b>13</b>) and <i>β</i>-sitosterol (<b>14</b>) were isolated from dichloromethane-soluble portion of <i>Polygonum hydropiper</i>. By using Alamar blue assay, compounds <b>2</b>, <b>7</b>, <b>8</b>, <b>11</b> and <b>12</b> were found to be active against <i>Trypanosoma brucei</i> with IC₅₀ values in the range of 0.49–7.77 μg/mL. Cardamomin (<b>11</b>) had most significant activity against <i>T. brucei</i> with IC₅₀/IC₉₀ values of 0.49/0.81 μg/mL compared to the positive control DFMO (IC₅₀/IC₉₀: 3.02/8.05 μg/mL). Furthermore, in antimalarial, antimicrobial, anti-inflammatory, PPAR and cytotoxic assays, some compounds have demonstrated moderate inhibitory potentials.</p

UPLC-MS-ELSD-PDA as a Powerful Dereplication Tool to Facilitate Compound Identification from Small-Molecule Natural Product Libraries

The generation of natural product libraries containing column fractions, each with only a few small molecules, using a high-throughput, automated fractionation system, has made it possible to implement an improved dereplication strategy for selection and prioritization of leads in a natural product discovery program. Analysis of databased UPLC-MS-ELSD-PDA information of three leads from a biological screen employing the ependymoma cell line EphB2-EPD generated details on the possible structures of active compounds present. The procedure allows the rapid identification of known compounds and guides the isolation of unknown compounds of interest. Three previously known flavanone-type compounds, homoeriodictyol (<b>1</b>), hesperetin (<b>2</b>), and sterubin (<b>3</b>), were identified in a selected fraction derived from the leaves of <i>Eriodictyon angustifolium</i>. The lignan compound deoxypodophyllotoxin (<b>8</b>) was confirmed to be an active constituent in two lead fractions derived from the bark and leaves of <i>Thuja occidentalis</i>. In addition, two new but inactive labdane-type diterpenoids with an uncommon triol side chain were also identified as coexisting with deoxypodophyllotoxin in a lead fraction from the bark of <i>T. occidentalis.</i> Both diterpenoids were isolated in acetylated form, and their structures were determined as 14<i>S</i>,15-diacetoxy-13<i>R</i>-hydroxylabd-8­(17)-en-19-oic acid (<b>9</b>) and 14<i>R</i>,15-diacetoxy-13<i>S</i>-hydroxylabd-8­(17)-en-19-oic acid (<b>10</b>), respectively, by spectroscopic data interpretation and X-ray crystallography. This work demonstrates that a UPLC-MS-ELSD-PDA database produced during fractionation may be used as a powerful dereplication tool to facilitate compound identification from chromatographically tractable small-molecule natural product libraries