Development of a selective novel fluorescent substrate for sodium-dependent transporters.

Aim: To synthesize, characterize, and validate 6FGA, a fluorescent glucose modified with a Cyanine5.5 at carbon-6 position, for probing the function of sodium-dependent glucose transporters, SGLT1 and SGLT2.

Main Methods: The synthesis of fluorescent glucose analogue was achieved through "click chemistry" of Cyanine5.

Molecular Imaging of Tumors Using a Quantitative T 1 Mapping Technique via Magnetic Resonance Imaging.

Magnetic resonance imaging (MRI) of glioblastoma multiforme (GBM) with molecular imaging agents would allow for the specific localization of brain tumors. Prior studies using T 1-weighted MR imaging demonstrated that the SBK2-Tris-(Gd-DOTA)3 molecular imaging agent labeled heterotopic xenograft models of brain tumors more intensely than non-specific contrast agents using conventional T 1-weighted imaging techniques. In this study, we used a dynamic quantitative T 1 mapping strategy to more objectively compare intra-tumoral retention of the SBK2-Tris-(Gd-DOTA)3 agent over time in comparison to non-targeted control agents.

Peptide-Targeted Gold Nanoparticles for Photodynamic Therapy of Brain Cancer.

Targeted drug delivery using epidermal growth factor peptide-targeted gold nanoparticles (EGF-Au NPs) is investigated as a novel approach for delivery of photodynamic therapy (PDT) agents, specifically Pc 4, to cancer. In vitro studies of PDT show that EGF-Au NP-Pc 4 is twofold better at killing tumor cells than free Pc 4 after increasing localization in early endosomes. In vivo studies show that targeting with EGF-Au NP-Pc 4 improves accumulation of fluorescence of Pc 4 in subcutaneous tumors by greater than threefold compared with untargeted Au NPs.

An optical probe for noninvasive molecular imaging of orthotopic brain tumors overexpressing epidermal growth factor receptor.

We have developed a near-infrared (NIR) probe that targets cells overexpressing the EGF receptor (EGFR) for imaging glioblastoma brain tumors in live subjects. A peptide specific for the EGFR was modified with various lengths of monodiscrete polyethylene glycol (PEG) units and a NIR Cy5.5 fluorescence dye.

Addressing brain tumors with targeted gold nanoparticles: a new gold standard for hydrophobic drug delivery?

EGF-modified Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor compared to untargeted conjugates. The hydrophobic photodynamic therapy drug Pc 4 can be delivered efficiently into glioma brain tumors by EGF peptide-targeted Au NPs. Compared to the untargeted conjugates, EGF-Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor.

Novel peptide ligands with dual acting pharmacophores designed for the pathophysiology of neuropathic pain.

The conventional design of high affinity drugs targeted to a single molecule has not resulted in clinically useful therapies for pain relief. Recent reviews have suggested that newly designed analgesic drugs should incorporate multiple targets. The distributions of cholecystokinin (CCK) and CCK receptors in the central nervous system (CNS) overlap significantly with endogenous opioid systems and can be dually targeted.

Suborganelle sensing of mitochondrial cAMP-dependent protein kinase activity.

A fluorescent sensor of protein kinase activity has been developed and used to characterize the compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher with Arg residues positioned on the peptide substrate.

Neuropathic pain is maintained by brainstem neurons co-expressing opioid and cholecystokinin receptors.

Descending input from the rostral ventromedial medulla (RVM) provides positive and negative modulation of spinal nociceptive transmission and has been proposed to be critical for maintaining neuropathic pain. This study tests the hypothesis that neuropathic pain requires the activity of a subset of RVM neurons that are distinguished by co-expression of mu opioid receptor (MOR) and cholecystokinin type 2 receptor (CCK2). Using male Sprague-Dawley rats, we demonstrate that discrete RVM neurons express MOR and CCK2; over 80% of these cells co-express both receptors.

Structure-activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors.

Prolonged opioid exposure increases the expression of cholecystokinin (CCK) and its receptors in the central nervous system (CNS), where CCK may attenuate the antinociceptive effects of opioids. The complex interactions between opioid and CCK may play a role in the development of opioid tolerance. We designed and synthesized cyclic disulfide peptides and determined their agonist properties at opioid receptors and antagonist properties at CCK receptors.

Opioid and melanocortin receptors: do they have overlapping pharmacophores?

We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the delta opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the delta opioid receptor (Ki = 0.

Photochemically-activated probes of protein-protein interactions.

The activity of light-activatable ("caged") compounds can be temporally and spatially controlled, thereby providing a means to interrogate intracellular biochemical pathways as a function of time and space. Nearly all caged peptides contain photocleavable groups positioned on the side chains of key residues. We describe an alternative active site targeted strategy that disrupts the interaction between the protein target (SH2 domain, kinase, and proteinase) and a critical amide NH moiety of the peptide probe.

Understanding the structural requirements of 4-anilidopiperidine analogues for biological activities at mu and delta opioid receptors.

New 4-anilidopiperidine analogues in which the phenethyl group of fentanyl was replaced by several aromatic ring-contained amino acids (or acids) were synthesized to study the biological effect of the substituents on mu and delta opioid receptor interactions. These analogues showed broad (47 nM-76 microM) but selective (up to 17-fold) binding affinities at the mu opioid receptor over the delta opioid receptor, as predicted from the message-address concept.

Deep quench: an expanded dynamic range for protein kinase sensors.

Assays that furnish a fluorescent readout of protein kinase activity provide a means to identify and characterize inhibitory agents, assess structure-function relationships, and correlate enzyme activity with cellular behavior. Although several protein kinase sensors have been described in the literature, their fluorescent response to phosphorylation are generally modest to moderate (1.1 – 8-fold).

Partial retro-inverso, retro, and inverso modifications of hydrazide linked bifunctional peptides for opioid and cholecystokinin (CCK) receptors.

Partially modified retro-inverso, retro, and inverso isomers of hydrazide linked bifunctional peptides were designed, synthesized, and evaluated for bioactivities at delta/mu opioid receptors and CCK-1/CCK-2 receptors. All modifications of the CCK pharmacophore moiety affected bioactivities for the CCK-1 and CCK-2 receptors (up to 180-fold increase in the binding affinity with higher selectivity) and for the delta and mu opioid receptors. The results indicate that the opioid and CCK pharmacophores in one molecule interact with each other to induce topographical changes for both pharmacophores.

New paradigms and tools in drug design for pain and addiction.

New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at mu and delta opioid receptors and antagonist activities at cholecystokinin (CCK) receptors.

Structure-activity relationships of bifunctional peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors.

Cholecystokinin (CCK) has been identified as a pronociceptive endogenous peptide which also possesses antiopioid actions. CCK may be upregulated in conditions of chronic pain or during sustained morphine administration resulting in attenuation of opioid-mediated pain relief. These complex interactions between opioids and endogenous CCK receptor systems have suggested the need for a new paradigm in drug design for some states of chronic pain.

Design and synthesis of novel hydrazide-linked bifunctional peptides as delta/mu opioid receptor agonists and CCK-1/CCK-2 receptor antagonists.

A series of hydrazide-linked bifunctional peptides designed to act as agonists for delta/mu opioid receptors and antagonists for CCK-1/CCK-2 receptors was prepared and tested for binding to both opioid and CCK receptors and in functional assays. SAR studies in the CCK region examined the structural requirements for the side chain groups at positions 1', 2', and 4' and for the N-terminal protecting group, which are related to interactions not only with CCK, but also with opioid receptors. Most peptide ligands that showed high binding affinities (0.

Novel design of bicyclic beta-turn dipeptides on solid-phase supports and synthesis of [3.3.0]-Bicyclo([2,3])-leu-enkephalin analogues.

[structure: see text] External bicyclic beta-turn dipeptide mimetics provide an excellent design approach that can offer a rich chiral ensemble of structures with different backbone conformations. We report herein a novel design of a convergent combinatorial synthetic methodology, which is illustrated by the solid-phase synthesis of a series of [3.3.