Defining modulatory inputs into CNS neuronal subclasses by functional pharmacological profiling.

Previously we defined neuronal subclasses within the mouse peripheral nervous system using an experimental strategy called "constellation pharmacology." Here we demonstrate the broad applicability of constellation pharmacology by extending it to the CNS and specifically to the ventral respiratory column (VRC) of mouse brainstem, a region containing the neuronal network controlling respiratory rhythm. Analysis of dissociated cells from this locus revealed three major cell classes, each encompassing multiple subclasses. We broadly analyzed the combinations (constellations) of receptors and ion channels expressed within VRC cell classes and subclasses. These were strikingly different from the constellations of receptors and ion channels found in subclasses of peripheral neurons from mouse dorsal root ganglia. Within the VRC cell population, a subset of dissociated neurons responded to substance P, putatively corresponding to inspiratory pre-Bötzinger complex (preBötC) neurons. Using constellation pharmacology, we found that these substance P-responsive neurons also responded to histamine, and about half responded to bradykinin. Electrophysiological studies conducted in brainstem slices confirmed that preBötC neurons responsive to substance P exhibited similar responsiveness to bradykinin and histamine. The results demonstrate the predictive utility of constellation pharmacology for defining modulatory inputs into specific neuronal subclasses within central neuronal networks.

From molecular phylogeny towards differentiating pharmacology for NMDA receptor subtypes.

In order to decode the roles that N-methyl-D-aspartate (NMDA) receptors play in excitatory neurotransmission, synaptic plasticity, and neuropathologies, there is need for ligands that differ in their subtype selectivity. The conantokin family of Conus peptides is the only group of peptidic natural products known to target NMDA receptors. Using a search that was guided by phylogeny, we identified new conantokins from the marine snail Conus bocki that complement the current repertoire of NMDA receptor pharmacology. Channel currents measured in Xenopus oocytes demonstrate conantokins conBk-A, conBk-B, and conBk-C have highest potencies for NR2D containing receptors, in contrast to previously characterized conantokins that preferentially block NR2B containing NMDA receptors. Conantokins are rich in γ-carboxyglutamate, typically 17-34 residues, and adopt helical structure in a calcium-dependent manner. As judged by CD spectroscopy, conBk-C adopts significant helical structure in a calcium ion-dependent manner, while calcium, on its own, appears insufficient to stabilize helical conformations of conBk-A or conBk-B. Molecular dynamics simulations help explain the differences in calcium-stabilized structures. Two-dimensional NMR spectroscopy shows that the 9-residue conBk-B is relatively unstructured but forms a helix in the presence of TFE and calcium ions that is similar to other conantokin structures. These newly discovered conantokins hold promise that further exploration of small peptidic antagonists will lead to a set of pharmacological tools that can be used to characterize the role of NMDA receptors in nervous system function and disease.

Conantokins derived from the Asprella clade impart conRl-B, an N-methyl d-aspartate receptor antagonist with a unique selectivity profile for NR2B subunits.

Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-methyl d-aspartate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity with the most studied NR2B selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogues for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings for Tyr5 substitutions in other conantokins, conRl-B[L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis, and pharmacology for discovering new bioactive natural products.

Characterization of conantokin Rl-A: molecular phylogeny as structure/function study.

A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl-A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl-A is a 24-residue peptide containing three gamma-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl-A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl-A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca(++). The order of potency for blocking the four NMDA receptor subtypes by ConRl-A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl-A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus.

Neuroprotective and cardioprotective conopeptides: an emerging class of drug leads.

The peptides in the venoms of predatory marine snails belonging to the genus Conus ('cone snails') have well-established therapeutic applications for the treatment of pain and epilepsy. This review discusses the neuroprotective and cardioprotective potential of four families of Conus peptides (conopeptides), including omega-conotoxins that target voltage-gated Ca2+ channels, conantokins that target NMDA receptors, mu-conotoxins that target voltage-gated Na+ channels, and kappa- and kappaM-conotoxins that target K+ channels. The diversity of Conus peptides that have already been shown to exhibit neuroprotective/cardioprotective activity suggests that marine snail venoms are a potentially rich source of drug leads with diverse mechanisms.

Conantokin-Br from Conus brettinghami and selectivity determinants for the NR2D subunit of the NMDA receptor.

Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami Coomans , H. E. , Moolenbeek , R. G. and Wils , E. ( 1982 ) Basteria 46 ( 1/4 ), 3 - 67 , conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2-13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R); the latter has a approximately 30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9-10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogues comprising 1-3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.

Conantokin-P, an unusual conantokin with a long disulfide loop.

The conantokins are a family of Conus venom peptides (17-27AA) that are N-methyl-d-aspartate (NMDA) receptor antagonists. Conantokins lack disulfide bridges (six out of seven previously characterized peptides are linear), but contain multiple residues of gamma-carboxyglutamate. These post-translationally modified amino acids confer the largely helical structure of conantokins by coordinating divalent metal ions. Here, we report that a group of fish-hunting cone snails, Conus purpurascens and Conus ermineus, express a distinctive branch of the conantokin family in their venom ducts. Two novel conantokins, conantokin-P (Con-P) and conantokin-E (Con-E) are 24AA long and contain five gamma-carboxyglutamate residues. These two peptides are characterized by a long disulfide loop (12 amino acids including two Gla residues between the Cys residues). The oxidative folding studies of Con-P revealed that the formation of the disulfide bond proceeded significantly faster in the presence of Ca(++) ions. Circular dichroism suggested that Con-P is less helical than other previously characterized conantokins. Con-P blocks NMDA receptors containing NR2B subunit with submicromolar potency. Furthermore, the subtype-selectivity for different NR2 subunits differs from that of the previously characterized conantokins. Our results suggest that different branches of the phylogenetic tree of cone snails have evolved distinct groups of conantokins, each with its own unique biochemical features.

Novel conantokins from Conus parius venom are specific antagonists of N-methyl-D-aspartate receptors.

We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2, and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a post-translationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the 3rd position from the N terminus, where the Gla residue is replaced by either aspartate or by another post-translationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different post-translational modifications. Conantokins-Pr1 and -Pr2 adopt alpha-helical conformations in the presence of divalent cations (Mg2+ and Ca2+) but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-d-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2, and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.

The neurosteroids dehydroepiandrosterone sulfate and pregnenolone sulfate inhibit the UNC-49 GABA receptor through a common set of residues.

Neurosteroids are endogenous neuromodulators that bind and allosterically regulate GABA(A) receptors. Residues were recently identified in the first transmembrane domain (M1) of GABA(A) receptor subunits that are important for neurosteroid modulation. We are studying the inhibition of GABA(A) receptors by sulfated neurosteroids. One of these neurosteroid, pregnenolone sulfate (PS), depends on six identified M1 residues to inhibit the UNC-49 GABA receptor, a homomeric GABA receptor from Caenorhabditis elegans that is homologous to the mammalian GABA(A) receptor. Here, we investigate the inhibition of the UNC-49 GABA receptor by another sulfated neurosteroid, dehydroepiandrosterone sulfate (DHEAS). DHEAS is identical to PS except that it contains a carbonyl oxygen instead of an acetyl group at C17 on the steroid D ring. UNC-49 mutations that affect PS inhibition had broadly parallel effects on DHEAS, suggesting the two neurosteroids act through similar mechanisms. However, certain M1 mutations affected DHEAS differently than PS. Considering that first, the D ring contains the only structural difference between PS and DHEAS, and second, the strongest chemical and steric effects of a mutation are likely to be felt in the local environment of the altered residues, this result implies that the steroid D ring may contact M1 near the mutated residues. This possibility is interesting because current models of neurosteroid interactions with GABA(A) receptors, based on pregnane steroids, suggest that the steroid A ring binds M1, whereas the D ring binds M4. Our findings suggest that there may be considerable diversity in the way different classes of neurosteroids interact with GABA(A) receptors.