Palladium-Catalyzed Regio- and Stereoselective γ‑Arylation of Tertiary Allylic Amines: Identification of Potent Adenylyl Cyclase Inhibitors

Substituted allylic amines and their derivatives are key structural motifs of many drug molecules and natural products. A general, mild, and practical palladium-catalyzed γ-arylation of tertiary allylic amines, one of the most challenging Heck arylation substrates, has been developed. The γ-arylation products were obtained in excellent regio- and stereoselectivity. Moreover, novel and potent adenylyl cyclase inhibitors with the potential for treating neuropathic and inflammatory pain have been identified from the γ-arylation products

O6C-20-nor-SalA is a stable and potent KOR agonist

Salvinorin A (SalA) is a potent and selective agonist of the kappa-opioid receptor (KOR), but its instability has frustrated medicinal chemistry efforts. Treatment of SalA with weak bases like DBU leads to C8 epimerization with loss of receptor affinity and signaling potency. Here we show that replacement of C20 with H and replacement of O6 with CH2 stabilizes the SalA scaffold relative to its C8 epimer, so much so that epimerization is completely suppressed. This new compound, O6C-20-nor-SalA, retains high potency for agonism of KOR. <br

Polydopamine-Based Simple and Versatile Surface Modification of Polymeric Nano Drug Carriers

The surface of a polymeric nanoparticle (NP) is often functionalized with cell-interactive ligands and/or additional polymeric layers to control NP interaction with cells and proteins. However, such modification is not always straightforward when the surface is not chemically reactive. For this reason, most NP functionalization processes employ reactive linkers or coupling agents or involve prefunctionalization of the polymer, which are complicated and inefficient. Moreover, prefunctionalized polymers can lose the ability to encapsulate and retain a drug if the added ligands change the chemical properties of the polymer. To overcome this challenge, we use dopamine polymerization as a way of functionalizing NP surfaces. This method includes brief incubation of the preformed NPs in a weak alkaline solution of dopamine, followed by secondary incubation with desired ligands. Using this method, we have functionalized poly(lactic-<i>co</i>-glycolic acid) (PLGA) NPs with three representative surface modifiers: a small molecule (folate), a peptide (Arg-Gly-Asp), and a polymer [poly(carboxybetaine methacrylate)]. We confirmed that the modified NPs showed the expected cellular interactions with no cytotoxicity or residual bioactivity of dopamine. The dopamine polymerization method is a simple and versatile surface modification method, applicable to a variety of NP drug carriers irrespective of their chemical reactivity and the types of ligands

A “Genome-to-Lead” Approach for Insecticide Discovery: Pharmacological Characterization and Screening of <em>Aedes aegypti</em> D₁-like Dopamine Receptors

<div><h3>Background</h3><p>Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides.</p> <h3>Methodology/Principal Findings</h3><p>We describe a “genome-to-lead” approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (<em>Aa</em>DOP1 and <em>Aa</em>DOP2) from the yellow fever mosquito, <em>Aedes aegypti</em>. Sequence analyses indicated that these receptors are orthologous to arthropod D₁-like (Gα_s-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of <em>Aa</em>DOP1 and <em>Aa</em>DOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC₅₀: <em>Aa</em>DOP1 = 3.1±1.1 nM; <em>Aa</em>DOP2 = 240±16 nM). Interestingly, only <em>Aa</em>DOP1 exhibited sensitivity to epinephrine (EC₅₀ = 5.8±1.5 nM) and norepinephrine (EC₅₀ = 760±180 nM), while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at <em>Aa</em>DOP2. Fifty-one compounds were identified as “hits,” and follow-up validation assays confirmed the antagonistic effect of selected compounds at <em>Aa</em>DOP2. <em>In vitro</em> comparison studies between <em>Aa</em>DOP2 and the human D₁ dopamine receptor (hD₁) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as <em>Aa</em>DOP2-selective compounds. In subsequent <em>Ae. aegypti</em> larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as “leads” for insecticide discovery.</p> <h3>Conclusions/Significance</h3><p>This research provides a “proof-of-concept” for a novel approach toward insecticide discovery, in which genome sequence data are utilized for functional characterization and chemical compound screening of GPCRs. We provide a pipeline useful for future prioritization, pharmacological characterization, and expanded chemical screening of additional GPCRs in disease-vector arthropods. The differential molecular and pharmacological properties of the mosquito dopamine receptors highlight the potential for the identification of target-specific chemistries for vector-borne disease management, and we report the first study to identify dopamine receptor antagonists with <em>in vivo</em> toxicity toward mosquitoes.</p> </div

Confirmation and secondary assays for “hit” antagonists of <i>Aa</i>DOP2 and human D₁ receptor.

<p>Select chemistries and the assay control (SCH23390) were tested in dose-response cAMP assays in the presence of 3 µM dopamine in <i>Aa</i>DOP2- or 100 nM dopamine in hD₁-expressing cells (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001478#pntd-0001478-g005" target="_blank">Figure 5</a>). Compounds with IC₅₀ values ≥10 µM are considered to lack activity at <i>Aa</i>DOP2 and were not tested at hD₁. N.D. = not determined; hD₁ = Human D₁ dopamine receptor.</p

Neighbor-joining sequence analysis of <i>Aedes aegypti Aa</i>DOP1 and AaDOP2 and representative biogenic amine receptors.

<p>The deduced amino acid sequences for the mosquito dopamine receptors <i>Aa</i>DOP1 and AaDOP2 and additional receptors for dopamine, muscarinic acetylcholine, octopamine, serotonin, and tyramine from <i>Drosophila melanogaster</i> and <i>Apis mellifera</i>, as well as the human D₁-like and D₂-like dopamine receptors were aligned for use in the analysis. Bootstrap values (100 replicates) are indicated with numbers at supported branches. The outgroup is a <i>D. melanogaster</i> diuretic hormone receptor, a Class B GPCR. Abbreviations: <i>Aa</i> = <i>Ae. aegypti</i>; <i>Is</i> = <i>I. scapularis</i>; <i>Dm</i> = <i>D. melanogaster</i>; <i>Am</i> = <i>A. mellifera</i>; <i>Hs</i> = <i>H. sapiens</i>. Sequences: <i>Is</i>dop1, D₁-like dopamine receptor (ISCW001496); <i>Is</i>dop2, D₁-like dopamine receptor (ISCW008775); <i>Dm</i>D-Dop1, D₁-like dopamine receptor (P41596); <i>Dm</i>DAMB, D₁-like dopamine receptor (DopR99B/DAMB: AAC47161), <i>Dm</i>DD2R, D₂-like dopamine receptor (DD2R-606: AAN15955); <i>Dm</i>Dih, diuretic hormone 44 receptor 1 (NP_610960.1); <i>Dm</i>mAChR, muscarinic acetylcholine receptor (AAA28676); <i>Dm</i>OAMB, octopamine receptor in mushroom bodies, isoform A (NP_732541); DM5HT1A, serotonin receptor 1A, isoform A (NP_476802); <i>Dm</i>Tyr, tyramine receptor (CG7431: NP_650652); <i>Am</i>DOP1, D₁-like dopamine receptor (dopamine receptor, D1, NP_001011595); <i>Am</i>DOP2, D₁-like dopamine receptor (dopamine receptor 2, NP_001011567), <i>Am</i>DOP3, D₂-like dopamine receptor (<i>Am</i>DOP3, NP_001014983); <i>Am</i>mAChR, muscarinic acetylcholine receptor (XP_395760); <i>Am</i>OA1, octopamine receptor (oar, NP_001011565); <i>Am</i>5HT1A, serotonin receptor (5ht-1, NP_001164579); <i>Am</i>Tyr, tyramine receptor (XP_394231); <i>Hs</i>D1, D₁-like dopamine receptor (D(1A), NP_000785); <i>Hs</i>D2,D₂-like dopamine receptor (D(2), NP_000786); <i>Hs</i>D3, D₂-like dopamine receptor (D(3), NP_000787); <i>Hs</i>D4, D₂-like dopamine receptor (D(4), NP_000788); <i>Hs</i>D5, D₁-like dopamine receptor (D(1B)/D5, NP_000789).</p

Toxicity of antagonist screen hits in <i>Ae. aegypti</i> larval bioassays.

<p><b>A: </b><i>Ae. aegypti</i> larval bioassay showing toxicity of amitriptyline and doxepin at a single dose point (400 µM) compared to the water control; Ami = amitriptyline, Dox = Doxepin; * indicates <i>p</i><0.05; <b>B: </b><i>Ae. aegypti</i> larval bioassay involving amitriptyline in a dose-response format (25 µM–400 µM).</p

Alignment of the complete <i>Aedes aegypti Aa</i>DOP1 and <i>Aa</i>DOP2 amino acid sequences.

<p>Highlighted areas designate residues with shared biochemical characteristics, as designated by the ClustalW <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001478#pntd.0001478-Chenna1" target="_blank">[33]</a> output, where black shading = identical residues; dark shading = strongly similar residues; light shading = weakly similar residues. Also noted are the residues composing the N- and C-termini and the transmembrane (TM) domains I–VII.</p

Responses of <i>Aa</i>DOP1 and <i>Aa</i>DOP2 to biogenic amines and synthetic dopamine receptor agonists.

<p>HEK293 cells stably expressing both a CRELuc reporter construct and either of the receptors were stimulated with potential agonists. Dose-response curves were plotted and the EC₅₀ values were calculated. Compounds with EC₅₀ values ≥10 µM are considered to lack intrinsic activity at <i>Aa</i>DOP2.</p

Drug discovery and development pipeline for new insecticidal chemistries.

<p><b>A:</b> The illustration shows critical steps involved with the “genome-to-lead” (described in this manuscript) and “lead-to-product” phases. Abbreviations: (EPA) Environmental Protection Agency; (FDA) Food and Drug Administration; (SAR) structure-activity relationship study. The intended administration route of a particular chemistry dictates the federal agency that will receive the registration package; <b>B:</b> Expanded details of the “hit-to-lead” phase including those pursued in this study.</p