Using Constellation Pharmacology to Characterize a Novel α-Conotoxin from Conus ateralbus.

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3ß4, α6/α3ß4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3ß4 and α6/α3ß4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.

Nicotinic Acetylcholine Receptor Partial Antagonist Polyamides from Tunicates and Their Predatory Sea Slugs.

In our efforts to discover new drugs to treat pain, we identified molleamines A-E (1-5) as major neuroactive components of the sea slug, Pleurobranchus forskalii, and their prey, Didemnum molle, tunicates. The chemical structures of molleamines were elucidated by spectroscopy and confirmed by the total synthesis of molleamines A (1) and C (3). Synthetic 3 completely blocked acetylcholine-induced calcium flux in peptidergic nociceptors (PNs) in the somatosensory nervous system. Compound 3 affected neither the α7 nAChR nor the muscarinic acetylcholine receptors in calcium flux assays. In addition to nociceptors, 3 partially blocked the acetylcholine-induced calcium flux in the sympathetic nervous system, including neurons from the superior cervical ganglion. Electrophysiology revealed a block of α3ß4 (mouse) and α6/α3ß4 (rat) nicotinic acetylcholine receptors (nAChRs), with IC50 values of 1.4 and 3.1 µM, respectively. Molleamine C (3) is a partial antagonist, reaching a maximum block of 76-82% of the acetylcholine signal and showing no partial agonist response. Molleamine C (3) may thus provide a lead compound for the development of neuroactive compounds with unique biological properties.

Chronicling changes in the somatosensory neurons after peripheral nerve injury.

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain.

Synergistic anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and absolute stereochemistry of 7,8-dideoxygriseorhodin C.

The emergence of antibiotic resistance necessitates not only the identification of new compounds with antimicrobial properties, but also new strategies and combination therapies to circumvent this growing problem. Here, we report synergistic activity against methicillin-resistant Staphylococcus aureus (MRSA) of the ß-lactam antibiotic oxacillin combined with 7,8-dideoxygriseorhodin C in vitro. Ongoing efforts to identify antibiotics from marine mollusk-associated bacteria resulted in the isolation of 7,8-dideoxygriseorhodin C from a Streptomyces sp. strain cultivated from a marine gastropod tissue homogenate. Despite the long history of 7,8-dideoxygriseorhodin C in the literature, the absolute configuration has never been previously reported. A comparison of measured and calculated ECD spectra resolved the configuration of the spiroketal carbon C6, and 2D ROESY NMR spectroscopy established the absolute configuration as 6s,6aS. The compound is selective against Gram-positive bacteria including MRSA and Enterococcus faecium with an MIC range of 0.125-0.5 µg ml-1. Moreover, the compound synergizes with oxacillin against MRSA as observed in the antimicrobial microdilution and time-kill assays. Simultaneous treatment of the compound with oxacillin resulted in an approximately tenfold decrease in MIC with a combination index of <0.5, indicating synergistic anti-MRSA activity.

Inhibition of Biofilm Formation by Modified Oxylipins from the Shipworm Symbiont Teredinibacter turnerae.

The bioactivity-guided purification of the culture broth of the shipworm endosymbiont Teredinibacter turnerae strain 991H.S.0a.06 yielded a new fatty acid, turneroic acid (1), and two previously described oxylipins (2-3). Turneroic acid (1) is an 18-carbon fatty acid decorated by a hydroxy group and an epoxide ring. Compounds 1-3 inhibited bacterial biofilm formation in Staphylococcus epidermidis, while only 3 showed antimicrobial activity against planktonic S. epidermidis. Comparison of the bioactivity of 1-3 with structurally related compounds indicated the importance of the epoxide moiety for selective and potent biofilm inhibition.

Synergistic Cytotoxicity of Renieramycin M and Doxorubicin in MCF-7 Breast Cancer Cells.

Renieramycin M (RM) is a KCN-stabilized tetrahydroisoquinoline purified from the blue sponge Xestospongia sp., with nanomolar IC50s against several cancer cell lines. Our goal is to evaluate its combination effects with doxorubicin (DOX) in estrogen receptor positive MCF-7 breast cancer cells. MCF-7 cells were treated simultaneously or sequentially with various combination ratios of RM and DOX for 72 h. Cell viability was determined using the MTT assay. Synergism or antagonism was determined using curve-shift analysis, combination index method and isobologram analysis. Synergism was observed with pharmacologically achievable concentrations of DOX when administered simultaneously, but not sequentially. The IC95 values of RM and DOX after combination were reduced by up to four-fold and eight-fold, respectively. To gain insights on the mechanism of synergy, real-time profiling, cell cycle analysis, apoptosis assays, and transcriptome analysis were conducted. The combination treatment displayed a similar profile with DNA-damaging agents and induced a greater and faster cell killing. The combination treatment also showed an increase in apoptosis. DOX induced S and G2/M arrest while RM did not induce significant changes in the cell cycle. DNA replication and repair genes were downregulated commonly by RM and DOX. p53 signaling and cell cycle checkpoints were regulated by DOX while ErbB/PI3K-Akt, integrin and focal adhesion signaling were regulated by RM upon combination. Genes involved in cytochrome C release and interferon gamma signaling were regulated specifically in the combination treatment. This study serves as a basis for in vivo studies and provides a rationale for using RM in combination with other anticancer drugs.

Mindapyrroles A-C, Pyoluteorin Analogues from a Shipworm-Associated Bacterium.

Three new pyoluteorin analogues, mindapyrroles A-C (1-3), were purified from Pseudomonas aeruginosa strain 1682U.R.0a.27, a gill-associated bacterium isolated from the tissue homogenate of the giant shipworm Kuphus polythalamius. Mindapyrroles B and C inhibit the growth of multiple pathogenic bacteria, with mindapyrrole B (2) showing the most potent antimicrobial activity and widest selectivity index over mammalian cells. Preliminary structure-activity relationship analysis showed that dimerization of the pyoluteorin moiety through a C-C linkage is detrimental to the antimicrobial activity, but addition of an aerugine unit in the methylene bridge is favorable for both the antimicrobial activity and selectivity index.

On the inhibition of capsaicin response in dorsal root ganglion neurons by nobilamide B and analogues: a structure-activity relationship study.

Nobilamide B, a TRPV1 antagonist, and a series of Ala-substituted analogues were synthesized and their neuroactivity was assessed in a primary culture of dorsal root ganglion (DRG) neurons. Analogues 4, 6, and 7 showed comparable activity, affecting capsaicin response in neurons, in contrast to 2, 3, and 5 which showed minimal blocking. Compounds 2, 3, and 5 correspond to analogues in which d-Phe, d-Leu and d-allo-Thr have been substituted with Ala, respectively. The observed loss of bioactivity in these three analogues highlights the importance of d amino acids in the primary structure of nobilamide B.