PhID: An Open-Access Integrated Pharmacology Interactions Database for Drugs, Targets, Diseases, Genes, Side-Effects, and Pathways

The current network pharmacology study encountered a bottleneck with a lot of public data scattered in different databases. There is a lack of an open-access and consolidated platform that integrates this information for systemic research. To address this issue, we have developed PhID, an integrated pharmacology database which integrates >400 000 pharmacology elements (drug, target, disease, gene, side-effect, and pathway) and >200 000 element interactions in branches of public databases. PhID has three major applications: (1) assisting scientists searching through the overwhelming amount of pharmacology element interaction data by names, public IDs, molecule structures, or molecular substructures; (2) helping visualizing pharmacology elements and their interactions with a web-based network graph; and (3) providing prediction of drug–target interactions through two modules: PreDPI-ki and FIM, by which users can predict drug–target interactions of PhID entities or some drug–target pairs of their own interest. To get a systems-level understanding of drug action and disease complexity, PhID as a network pharmacology tool was established from the perspective of data layer, visualization layer, and prediction model layer to present information untapped by current databases

Essential Role of the Donor Acyl Carrier Protein in Stereoselective Chain Translocation to a Fully Reducing Module of the Nanchangmycin Polyketide Synthase

Incubation of recombinant module 2 of the polyether nanchangmycin synthase (NANS), carrying an appended thioesterase domain, with the ACP-bound substrate (2<i>RS</i>)-2-methyl-3-ketobutyryl-NANS_ACP1 (<b>2-ACP1</b>) and methylmalonyl-CoA in the presence of NADPH gave diastereomerically pure (2<i>S</i>,4<i>R</i>)-2,4-dimethyl-5-ketohexanoic acid (<b>4a</b>). These results contrast with the previously reported weak discrimination by NANS module 2+TE between the enantiomers of the corresponding <i>N</i>-acetylcysteamine-conjugated substrate analogue (±)-2-methyl-3-ketobutyryl-SNAC (<b>2-SNAC</b>), which resulted in formation of a 5:3 mixture of <b>4a</b> and its (2<i>S</i>,4<i>S</i>)-diastereomer <b>4b</b>. Incubation of NANS module 2+TE with <b>2-ACP1</b> in the absence of NADPH gave unreduced 3,5,6-trimethyl-4-hydroxypyrone (<b>3</b>) with a <i>k</i>_cat of 4.4 ± 0.9 min^–1 and a <i>k</i>_cat/<i>K</i>_m of 67 min^–1 mM^–1, corresponding to a ∼2300-fold increase compared to the <i>k</i>_cat/<i>K</i>_m for the diffusive substrate <b>2-SNAC</b>. Covalent tethering of the 2-methyl-3-ketobutyryl thioester substrate to the NANS ACP1 domain derived from the natural upstream PKS module of the nanchangmycin synthase significantly enhanced both the stereospecificity and the kinetic efficiency of the sequential polyketide chain translocation and condensation reactions catalyzed by the ketosynthase domain of NANS module 2

Mining of the Pyrrolamide Antibiotics Analogs in <i>Streptomyces netropsis</i> Reveals the Amidohydrolase-Dependent “Iterative Strategy” Underlying the Pyrrole Polymerization

<div><p>In biosynthesis of natural products, potential intermediates or analogs of a particular compound in the crude extracts are commonly overlooked in routine assays due to their low concentration, limited structural information, or because of their insignificant bio-activities. This may lead into an incomplete and even an incorrect biosynthetic pathway for the target molecule. Here we applied multiple compound mining approaches, including genome scanning and precursor ion scan-directed mass spectrometry, to identify potential pyrrolamide compounds in the fermentation culture of <i>Streptomyces netropsis</i>. Several novel congocidine and distamycin analogs were thus detected and characterized. A more reasonable route for the biosynthesis of pyrrolamides was proposed based on the structures of these newly discovered compounds, as well as the functional characterization of several key biosynthetic genes of pyrrolamides. Collectively, our results implied an unusual “iterative strategy” underlying the pyrrole polymerization in the biosynthesis of pyrrolamide antibiotics.</p></div

In-frame deletion of <i>pya25</i> and <i>pya26</i> in <i>S. netropsis</i>.

<p>HPLC analysis of pyrrolamides production in <i>S. netropsis</i> wild-type strain, the mutant strains WDY002 (Δ<i>pya25</i>) and WDY003 (Δ<i>pya26</i>), and the complementation strains WDY004 (negative control) and WDY005. Congocidine, Compound <b>3</b>, and Distamycin are indicated. The characteristic absorbance wave-length for pyrrolamides is 297 nm.</p

Identification of Congocidine (1), Distamycin (2), and a novel pyrrolamide compound (3) in <i>S. netropsis</i>.

<p>(A) High resolution mass spectrum of Congocidine and Distamycin. (B) Precursor ion scan-directed mass spectrum to identify compound <b>3</b>. Base peak chromatograms of precursor ion scan are shown. Ions of <i>m</i>/<i>z</i> 273 and 247 are daughter ions of compound <b>3</b>, and were used as the queries.</p

Organization of the pyrrolamides biosynthesis-related genes identified from <i>S. ambofaciens</i> (a congocidine producer) and <i>S. netropsis</i>.

<p>The deduced functions of each gene are summarized in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099077#pone.0099077.s001" target="_blank">File S1</a>. Homologies in sequence are indicated by plain and dashed lines (the latter pattern is for the separate gene cluster).</p

Illustration of the “iterative strategy” underlying pyrrolamide biosynthesis.

<p>The putative amidohydrolase Pya25 catalyzed the deacetylation of PCP-tethered pyrrolamide biosynthesis intermediates and determined the number of the pyrrole groups assembled into various pyrrolamides. A, adenylation domain; C, condensation domain; PCP, peptidyl carrier protein.</p

In vitro assays of the <i>Streptomyces coelicolor</i> AfsR SARP.

(A) Core promoter sequences used for in vitro assays. The mutations introduced into the afs box are highlighted. The mutated target sites contained mutations in upstream repeat (M1), the downstream repeat (M2), or both repeats (M1M2). The actII-4 promoter was used as a control. (B) Fluorescence polarization assays of the SARP with mutant afs box (M1, M2, and M1M2). Error bars represent mean ± SEM of n = 3 experiments. (C) In vitro MangoIII-based transcription assays with or without 500 nM SARP in the absence of RbpA and CarD. Error bars represent mean ± SEM of n = 3 experiments. The data underlying B and C can be found in S1 Data. (TIF)</p

Numerical values used to generate graphs.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the −35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β′ zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.</div

SARP interacts with σ^HrdB R4, β FTH, β′ ZBD.

(A) The SARP protomers interact with σHrdB, β, and β′. (B) The upstream SARP protomer contacts σHrdB R4 by its ODB domain. Salt bridges are shown as red dashed lines. (C) The H499 of σHrdBR4 is enfolded in an amphiphilic pocket of the BTA domain. The K496 of σHrdBR4 makes a salt bridge with the E246 of the BTA domain. (D) The downstream SARP protomer make extensive interactions with the β FTH, the preceding loop (TPL) and the following loop (TFL) of the β flap. SARP is colored orange and β flap is colored blue. Hydrogen bonds, salt-bridges, and van der Waals interactions are shown as yellow, red, and gray dashed lines, respectively. (E) Interactions between the β′ ZBD and the ODB of downstream SARP. The positively charged R67 and R69 of β′ ZBD contact the negatively charged E76 and E77 of the HTH loop of the ODB domain. (F) Mutating interfacial residues of SARP impaired transcription activation. The data underlying this figure can be found in S1 Data; error bars, SEM; n = 3; *P P Streptomyces strains, highlighting the residues interacting with β (blue), σ R4 (cyan), and αCTD (purple). The black boxes highlight the positions conserved. BTA, bacterial transcriptional activation; FTH, flap tip helix; ODB, OmpR-type DNA-binding; SARP, Streptomyces antibiotic regulatory protein; ZBD, zinc-binding domain.</p