We hypothesized that under chronic pain conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) in the spinal cord to promote hyperalgesia through an excitatory effect, which is opposite to the well known inhibitory effect of opioid receptors. Considering the structural dissimilarity between Dyn A and endogenous BR ligands, bradykinin (BK) and kallidin (KD), this interaction could not be predicted, but allowed us to discover a potential neuroexcitatory target. Well known BR ligands, BK, DALKD, and HOE140 showed different binding profiles at rat brain BRs than that previously reported. These results suggest that neuronal BRs in the rat central nervous system (CNS) may be pharmacologically distinct from those previously defined in non-neuronal tissues. Systematic structure-activity relationship (SAR) study at the rat brain BRs was performed and as a result, a new key structural feature of Dyn A for BR recognition was identified: amphipathicity. NMR studies of two lead ligands, Dyn A-(4-11) 7 and [des-Arg7]-Dyn A-(4-11) 14, which showed the same high binding affinity, confirmed that the Arg residue in position 7, which is known to be crucial for Dyn A’s biological activity, is not necessary, and that a type I β-turn structure at the C-terminal part of both ligands plays an important role in retaining good binding affinities at the BRs. Our lead ligand 14 blocked Dyn A-(2-13) 10-induced hyperalgesic effects and motor impairment in in vivo assays using naïve rats. In a model of peripheral neuropathy, intrathecal (i.th.) administration of ligand 14 reversed thermal hyperalgesia and mechanical hypersensitivity in a dose-dependent manner in nerve-injured rats. Thus ligand 14 may inhibit abnormal pain states by blocking the neuroexcitatory effects of enhanced levels of Dyn A, which are likely to be mediated by BRs in the spinal cord.

The design of artificial functional DNA-binding proteins has long been a goal for several research laboratories. The zinc finger proteins, which typically contain many fingers linked in tandem fashion, are some of the most studied DNA-binding proteins. The zinc finger protein's tandem arrangement and its the ability to recognize a wide variety of DNA sequences make it an attractive framework to design novel DNA-binding peptides/proteins. Our laboratory has utilized several design strategies to create novel zinc finger peptides by re-engineering the C(2)H(2)-type zinc finger motif of transcription factor Sp1. Some of the engineered zinc fingers have shown nuclease and catalytic functional properties. Based on these results, we present the design strategies for the creation of novel zinc fingers.

The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear. Host specificity and mutagenesis studies suggest that the helix D region of CD81 mediates binding to HCV-E2. Structural analysis of CD81 has enabled the synthesis of small molecules designed to mimic the space and hydrophobic features of the solvent-exposed face on helix D. Utilizing a novel bis-imidazole scaffold a series of over 100 compounds has been synthesized. Seven related, imidazole-based compounds were identified that inhibit binding of HCV-E2 to CD81. The inhibitory compounds have no short-term effect on cellular expression of CD81 or other tetraspanins, do not disrupt CD81 associations with other cell surface proteins, and bind reversibly to HCV-E2. These results provide an important proof of concept that CD81-based mimics can disrupt binding of HCV-E2 to CD81.

Development of opioid peptides as therapeutic agents has historically been limited due to pharmacokinetic issues including stability and blood-brain barrier (BBB) permeability. Glycosylation of opioid peptides can increase peptide serum stability and BBB penetration. To further define the requirements for optimizing in vivo antinociceptive potency following intravenous administration, we synthesized a series of enkephalin-based glycopeptides using solid phase 9-fluorenylmethyloxy carbamate methods. The compounds differed in the sixth and subsequent amino acid residues (Ser or Thr) and in the attached carbohydrate moiety. In vitro binding and functional smooth muscle bioassays indicated that the addition of mono-or disaccharides did not significantly affect the opioid receptor affinity or agonist activity of the glycopeptides compared with their unglycosylated parent peptides. All of the glycopeptides tested produced potent antinociceptive effects in male ICR mice following intracerebroventricular injection in the 55°C tail-flick test. The calculated A 50 values for the Ser/Thr and monosaccharide combinations were all very similar with values ranging from 0.02 to 0.09 nmol. Selected compounds were administered to mice intravenously and tested for antinociception to indirectly assess serum stability and BBB penetration. All compounds tested produced full antinociceptive effects with calculated A 50 values ranging from 2.2 to 46.4 mol/kg with the disaccharides having potencies that equaled or exceeded that of morphine on a micromoles per kilogram basis. Substitution of a trisaccharide or bis-and tris-monosaccharides resulted in a decrease in antinociceptive potency. These results provide additional support for the utility of glycosylation to increase central nervous system bioavailability of small peptides and compliment our ongoing stability and blood-brain barrier penetration studies.

Building on insights gained from the discovery of the antimalarial ozonide arterolane (OZ277), we now describe the structure-activity relationship (SAR) of the antimalarial ozonide artefenomel (OZ439). Primary and secondary amino ozonides had higher metabolic stabilities than tertiary amino ozonides, consistent with their higher pK and lower log D values. For primary amino ozonides, addition of polar functional groups decreased in vivo antimalarial efficacy. For secondary amino ozonides, additional functional groups had variable effects on metabolic stability and efficacy, but the most effective members of this series also had the highest log D values. For tertiary amino ozonides, addition of polar functional groups with H-bond donors increased metabolic stability but decreased in vivo antimalarial efficacy. Primary and tertiary amino ozonides with cycloalkyl and heterocycle substructures were superior to their acyclic counterparts. The high curative efficacy of these ozonides was most often associated with high and prolonged plasma exposure, but exposure on its own did not explain the presence or absence of either curative efficacy or in vivo toxicity.

Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood–brain barrier. Extremely potent and selective opiate agonists have been developed from endogenous peptides, some of which show great promise as drug candidates.

The application of endogenous neuropeptides (e.g., enkephalins) as analgesics has been retarded by their poor stability in vivo and by their inability to effectively penetrate the blood-brain barrier (BBB). Effective BBB transport of glycosylated enkephalins has been demonstrated in several labs now. Analgesia (antinociception) levels greater than morphine, and with reduced side effects have been observed for several glycopeptides related to enkephalin. Somewhat paradoxically, enhanced BBB transport across this lipophilic barrier is achieved by attaching water-soluble carbohydrate groups to the peptide moieties to produce biousian glycopeptides that can be either water-soluble or membrane bound. Transport is believed to rely on an endocytotic mechanism (transcytosis), and allows for systemic delivery and transport of the water-soluble glycopeptides. Much larger endorphin/dynorphin glycopeptide analogs bearing amphipathic helix address regions also have been shown to penetrate the BBB in mice. This holds forth the possibility of transporting much larger neuropeptides across the BBB, which may encompass a wide variety of receptors beyond the opioid receptors.

The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli the wild-type gene of the mature protein retains the methionine-0 which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli [R. E. Berry, T. K. Shokhireva, I. Filippov, M. N. Shokhirev, H. Zhang, F. A. Walker, Biochemistry 2007, 46, 6830]. Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different.