Thomas J. McMurry

Structural characterization of horseradish peroxidase using EXAFS spectroscopy. Evidence for Fe = O ligation in compounds I and II

Extended X-ray absorption fine structure spectroscopy has been utilized to determine the structural environment of the heme iron sites in horseradish peroxidase compounds I and II. For comparison, analogous studies have been undertaken on putative ferryl (Fe/sup IV/=O) porphyrin model compounds and on crystallographically characterized Cr/sup IV/=O and Cr/sup V/ identical with N porphyrins. In a preliminary communication, they suggested that a short ca. 1.6 A Fe-O bond is present in the high valent forms of both the enzyme and the synthetic porphyrins. The present work demonstrates unambiguously that a short, ca. 1.64 A, Fe-O bond length is present both in HRP compounds I and II and in their synthetic analogues. This structure is consistent only with an oxo-ferryl (Fe=O) complex as the active oxygen species in horseradish peroxidase. The structural details, their implications for heme protein mediated oxygen activation, and the difference between their results and those recently published by other workers.

EP-2104R: A Fibrin-Specific Gadolinium-Based MRI Contrast Agent for Detection of Thrombus

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.

Discovery of a CXCR4 agonist pepducin that mobilizes bone marrow hematopoietic cells

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341–mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.

Gadolinium Meets Medicinal Chemistry: MRI Contrast Agent Development

Magnetic resonance imaging (MRI) contrast agents are utilized adjunctively to enhance the contrast between normal and abnormal structures on MRI scans. Along with the rapid evolution of the field has come a new appreciation for the medicinal chemistry of this unique class of metallopharmaceuticals. The efficacy of MRI agents is a complex function of chemical, biophysical, and pharmacological properties, which must be married in a package of exquisite safety. This report illustrates the wide range of medicinal chemistry relevant to existing agents that are either approved or in clinical development, as well as concepts, which may result in exciting new pharmaceuticals in the future.

Direct Interaction between an Allosteric Agonist Pepducin and the Chemokine Receptor CXCR4

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.

An effective chelating agent for labelling of monoclonal antibody with 212Bi for α-particle mediated radioimmunotherapy

The ligand N-[2-amino-3-(p-isothiocyanatophenyl)propyl]-(±)-trans-1,2-diaminocyclohexane-N,N′,N″-pentaacetic acid has been synthesized and linked to IgG and to monoclonal antibody B72.3, and labelled with 206Bi and 212Bi to demonstrate the in vivo stability of the label and its utility for 212Bi-radioimmunotherapy.

Fibrin Specific Peptides Derived by Phage Display: Characterization of Peptides and Conjugates for Imaging

Peptides that bind to fibrin but not to fibrinogen or serum albumin were selected from phage display libraries as targeting moieties for thrombus molecular imaging probes. Three classes of cyclic peptides (cyclized via disulfide bond between two Cys) were identified with consensus sequences XArXCPY(G/D)LCArIX (Ar = aromatic, Tn6), X(2)CXYYGTCLX (Tn7), and NHGCYNSYGVPYCDYS (Tn10). These peptides bound to fibrin at ∼2 sites with K(d) = 4.1 μM, 4.0 μM, and 8.7 μM, respectively, whereas binding to fibrinogen was at least 100-fold weaker. The peptides also bind to the fibrin degradation product DD(E) with similar affinity to that measured for fibrin. The Tn7 and Tn10 peptides bind to the same site on fibrin, while the Tn6 peptides bind to a unique site. Alanine scanning identified the N- and C-terminal ends of the Tn6 and Tn7 peptides as most tolerant to modification. Peptide conjugates with either fluorescein or diethylenetriaminepentaaceto gadolinium(III) (GdDTPA) at the N-terminus were prepared for potential imaging applications, and these retained fibrin binding affinity and specificity in plasma. Relaxivity and binding studies on the GdDTPA derivatives revealed that an N-terminal glycyl linker had a modest effect on fibrin affinity but resulted in lower fibrin-bound relaxivity.

A direct synthesis of a bifunctional chelating agent for radiolabeling proteins

Abstract A convenient synthesis of a bifunctional derivative of the ligand NOTA is described. The synthesis offers advantages over current literature methods for making similar molecules by starting with the easily prepared ortho-amide of 1,4,7-triazacyclanonane which can be sequentially alkylated to provide a bifunctional NOTA derivative in 9% yield overall. The replacement of preparative HPLC with simple ion-exchange chromatography provides analytically pure ligand suitable for clinical applications.

Macrocyclic catechol-containing ligands

Tri-catechol ligands form selective and strong complexes with the ferric ion, making them attractive candidates for use as iron decorporation pharmaceuticals. Recent work in our group has concentrated on the synthesis of macrocyclic and macrobicyclic ligands incorporating the 2.3-dihydroxyterephthalamide binding subunit, which has been shown to be the most efficient bidentate iron chelator known (pMs ranging from 21.1 to 22.7). Macrocyclic polycatechol ligands with e ndocyclic b inding subunits were synthesized using high dilution techniques in yields of up to 24%. A series of macrobicyclic tri-catechol ligands in which the ligand properties were varied by incorporating TREN, mesitylene triamine, and TPT backbones, were synthesized in 10-15% yield via a stepwise approach employing high dilution conditions for the tripod-tripod coupling cyclization. In addition, a template synthesis was developed which allowed the preparation of (Et,NH),(Fe(bicapped TRENCAM)) in 50% overall yield. Reversible e lectrochemical behavior was observed above pH 11.5 for Na,(Fe(bicapped TRENCAM)) (E1/2 --.95V vs. NHE) and above pH 8.3 for Na3(Fe(6icapped TRENMECAM)) (E1/2 --.92 V. vs. NHE). The crystal structure of Na,(Fe(bicapped TRENCAM)) was determined and showed the coordination geometry about the metal center to be trigonal prismatic, an unprecedented geometry for Fe(II1).

Pepducins: lipopeptide allosteric modulators of GPCR signaling

Pepducins are a class of lipidated peptide G-protein coupled receptor (GPCR) allosteric modulators that exhibit unique pharmacological properties. Because of their rational design, pepducins can potentially be used to target all members of this gene family. In addition to enormous therapeutic potential, pepducins are proving to be valuable tools to elucidate the molecular mechanisms involved in GPCR signaling. In this review, we focus on the characterization of pepducin biology and discuss their utility as novel therapeutics.