Development of novel phenoxyalkylpiperidines as high-affinity Sigma-1 (σ) receptor ligands with potent anti-amnesic effect.

The sigma-1 (σ) receptor plays a significant role in many normal physiological functions and pathological disease states, and as such represents an attractive therapeutic target for both agonists and antagonists. Here, we describe a novel series of phenoxyalkylpiperidines based on the lead compound 1-[ω-(4-chlorophenoxy)ethyl]-4-methylpiperidine (1a) in which the degree of methylation at the carbon atoms alpha to the piperidine nitrogen was systematically varied. The affinity at σ and σ receptors and at Δ-Δ sterol isomerase (SI) ranged from subnanomolar to micromolar K values.

High-affinity sigma-1 (σ) receptor ligands based on the σ antagonist PB212.

The σ receptor is a druggable target involved in many physiological processes and diseases. To clarify its physiology and derive therapeutic benefit, nine analogs based on the σ antagonist PB212 were synthesized replacing the 4-methylpiperidine with basic moieties of varying size and degree of conformational freedom. 3-Phenylpyrrolidine, 4-phenylpiperidine or granatane derivatives displayed the highest affinity (.

"Methylene Bridge" to 5-HT Receptor Antagonists: Conformationally Constrained Phenylguanidines.

Arylguanidines, depending upon their aromatic substitution pattern, display varying actions at 5-HT receptors (e.g., partial agonist, agonist, superagonist).

Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40- Specific Endolysins.

Three bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity.

Complete Genome Sequences of Bacillus Phages Janet and OTooleKemple52.

We report here the genome sequences of two novel group-infecting bacteriophages, Janet and OTooleKemple52. These bacteriophages are double-stranded DNA-containing isolated from soil samples. While their genomes share a high degree of sequence identity with one another, their host preferences are unique.

Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody.

Maintaining pH-dependent conformational flexibility of M1 is critical for efficient influenza A virus replication.

The M gene segment of influenza A virus has been shown to be a contributing factor to the high growth phenotype. However, it remains largely unknown why matrix protein 1 (M1), the major structural protein encoded by M gene, exhibits pH-dependent conformational changes during virus replication. Understanding the mechanisms underlying efficient virus replication can help to develop strategies not only to combat influenza infections but also to improve vaccine supplies.

A Hexasaccharide Containing Rare 2-O-Sulfate-Glucuronic Acid Residues Selectively Activates Heparin Cofactor II.

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized.

Broad Spectrum Anti-Influenza Agents by Inhibiting Self-Association of Matrix Protein 1.

The matrix protein 1 (M1) of influenza A virus (IAV) exists as a three-dimensional oligomeric structure in mature virions with high sequence conservation across different IAV subtypes, which makes it a potential broad spectrum antiviral target. We hypothesized that impairing self-association of M1 through a small molecule 'wedge', which avidly binds to an M1-M1 interface, would result in a completely new class of anti-influenza agents. To establish this proof-of-principle, we performed virtual screening on a library of >70,000 commercially available small molecules that resulted in several plausible 'wedges'.

Superagonist, Full Agonist, Partial Agonist, and Antagonist Actions of Arylguanidines at 5-Hydroxytryptamine-3 (5-HT) Subunit A Receptors.

Introduction of minor variations to the substitution pattern of arylguanidine 5-hydroxytryptamine-3 (5-HT) receptor ligands resulted in a broad spectrum of functionally-active ligands from antagonist to superagonist. For example, (i) introduction of an additional Cl-substituent(s) to our lead full agonist N-(3-chlorophenyl)guanidine (mCPG, 2; efficacy % = 106) yielded superagonists 7-9 (efficacy % = 186, 139, and 129, respectively), (ii) a positional isomer of 2, p-Cl analog 11, displayed partial agonist actions (efficacy % = 12), and (iii) replacing the halogen atom at the meta or para position with an electron donating OCH group or a stronger electron withdrawing (i.e.

Estimating glycosaminoglycan-protein interaction affinity: water dominates the specific antithrombin-heparin interaction.

Glycosaminoglycan (GAG)-protein interactions modulate many important biological processes. Structure-function studies on GAGs may reveal probes and drugs, but their structural complexity and highly acidic nature confound such work. Productivity will increase if we are able to identify tight-binding oligosaccharides in silico.

Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions.

Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8-GAG interactions.

Single Amino Acid Variation Underlies Species-Specific Sensitivity to Amphibian Skin-Derived Opioid-like Peptides.

It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here, we test this idea by comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions.

A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior.

DREADDs are chemogenetic tools widely used to remotely control cellular signaling, neuronal activity, and behavior. Here we used a structure-based approach to develop a new Gi-coupled DREADD using the kappa-opioid receptor as a template (KORD) that is activated by the pharmacologically inert ligand salvinorin B (SALB). Activation of virally expressed KORD in several neuronal contexts robustly attenuated neuronal activity and modified behaviors.

Design, syntheses, and pharmacological characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan analogues as opioid receptor ligands.

A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1'- and/or 4'-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor.

Designing "high-affinity, high-specificity" glycosaminoglycan sequences through computerized modeling.

The prediction of high-affinity and/or high-specificity protein-glycosaminoglycan (GAG) interactions is an inherently difficult task, due to several factors including the shallow nature of the typical GAG-binding site and the inherent size, flexibility, diversity, and polydisperse nature of the GAG molecules. Here, we present a generally applicable methodology termed Combinatorial Library Virtual Screening (CVLS) that can identify potential high-affinity, high-specificity protein-GAG interactions from very large GAG combinatorial libraries and a suitable GAG-binding protein. We describe the CVLS approach along with the rationale behind it and provide validation for the method using the well-known antithrombin-thrombin-heparin system.

Crystal structures of influenza A virus matrix protein M1: variations on a theme.

Matrix protein 1 (M1) of the influenza A virus plays multiple roles in virion assembly and infection. Interest in the pH dependence of M1's multiple functions led us to study the effect of subtle pH changes on M1 structure, resulting in the elucidation of a unique low-pH crystal structure of the N(1-165)-domain of A/WSN/33 (H1N1) M1 that has never been reported. Although the 2.

Michael acceptor approach to the design of new salvinorin A-based high affinity ligands for the kappa-opioid receptor.

The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand-receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site.

Chemotype-selective modes of action of κ-opioid receptor agonists.

The crystal structures of opioid receptors provide a novel platform for inquiry into opioid receptor function. The molecular determinants for activation of the κ-opioid receptor (KOR) were studied using a combination of agonist docking, functional assays, and site-directed mutagenesis. Eighteen positions in the putative agonist binding site of KOR were selected and evaluated for their effects on receptor binding and activation by ligands representing four distinct chemotypes: the peptide dynorphin A(1-17), the arylacetamide U-69593, and the non-charged ligands salvinorin A and the octahydroisoquinolinone carboxamide 1xx.

Binding mode characterization of 6α- and 6β-N-heterocyclic substituted naltrexamine derivatives via docking in opioid receptor crystal structures and site-directed mutagenesis studies: application of the 'message-address' concept in development of mu opioid receptor selective antagonists.

Highly selective opioid receptor antagonists are essential pharmacological probes in opioid receptor structural characterization and opioid agonist functional studies. Currently, there is no highly selective, nonpeptidyl and reversible mu opioid receptor antagonist available. Among a series of naltrexamine derivatives that have been designed and synthesized, two compounds, NAP and NAQ, were previously identified as novel leads for this purpose based on their in vitro and in vivo pharmacological profiles.

On scaffold hopping: challenges in the discovery of sulfated small molecules as mimetics of glycosaminoglycans.

The design of sulfated, small, nonsaccharide molecules as modulators of proteins is still in its infancy as standard drug discovery tools such as library of diverse sulfated molecules and in silico docking and scoring protocol have not been firmly established. Databases, such as ZINC, contain too few sulfate-containing nonsaccharide molecules, which severely limits the identification of new hits. Lack of a generally applicable protocol for scaffold hopping limits the development of sulfated small molecules as synthetic mimetics of the highly sulfated glycosaminoglycans.

On the specificity of heparin/heparan sulfate binding to proteins. Anion-binding sites on antithrombin and thrombin are fundamentally different.

Background: The antithrombin-heparin/heparan sulfate (H/HS) and thrombin-H/HS interactions are recognized as prototypic specific and non-specific glycosaminoglycan (GAG)-protein interactions, respectively. The fundamental structural basis for the origin of specificity, or lack thereof, in these interactions remains unclear. The availability of multiple co-crystal structures facilitates a structural analysis that challenges the long-held belief that the GAG binding sites in antithrombin and thrombin are essentially similar with high solvent exposure and shallow surface characteristics.

The compensatory G88R change is essential in restoring the normal functions of influenza A/WSN/33 virus matrix protein 1 with a disrupted nuclear localization signal.

G88R emerged as a compensatory mutation in matrix protein 1 (M1) of influenza virus A/WSN/33 when its nuclear localization signal (NLS) was disrupted by R101S and R105S substitutions. The resultant M1 triple mutant M(NLS-88R) regained replication efficiency in vitro while remaining attenuated in vivo with the potential of being a live vaccine candidate. To understand why G88R was favored by the virus as a compensatory change for the NLS loss and resultant replication deficiency, three more M1 triple mutants with an alternative G88K, G88V, or G88E change in addition to R101S and R105S substitutions in the NLS were generated.

Designing allosteric regulators of thrombin. Monosulfated benzofuran dimers selectively interact with Arg173 of exosite 2 to induce inhibition.

Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. J. Med.