Charge and hydrophobicity effects of NIR fluorophores on bone-specific imaging.

Recent advances in near-infrared (NIR) fluorescence imaging enabled real-time intraoperative detection of bone metastases, bone growth, and tissue microcalcification. Pamidronate (PAM) has been widely used for this purpose because of its high binding affinity toward bone and remarkable therapeutic effects. Herein we describe the development of a series of PAM-conjugated NIR fluorophores that varied in net charges and hydrophobicity, and compared their bone targeting efficiency, biodistribution, and blood clearance. Since the targeting moiety, PAM, is highly negatively charged but small, the overall in vivo bone targeting and biodistribution were mediated by the physicochemical properties of conjugated fluorophores.

Structure-activity relationship of nerve-highlighting fluorophores.

Nerve damage is a major morbidity associated with numerous surgical interventions. Yet, nerve visualization continues to challenge even the most experienced surgeons. A nerve-specific fluorescent contrast agent, especially one with near-infrared (NIR) absorption and emission, would be of immediate benefit to patients and surgeons. Currently, there are only three classes of small molecule organic fluorophores that penetrate the blood nerve barrier and bind to nerve tissue when administered systemically. Of these three classes, the distyrylbenzenes (DSBs) are particularly attractive for further study. Although not presently in the NIR range, DSB fluorophores highlight all nerve tissue in mice, rats, and pigs after intravenous administration. The purpose of the current study was to define the pharmacophore responsible for nerve-specific uptake and retention, which would enable future molecules to be optimized for NIR optical properties. Structural analogs of the DSB class of small molecules were synthesized using combinatorial solid phase synthesis and commercially available building blocks, which yielded more than 200 unique DSB fluorophores. The nerve-specific properties of all DSB analogs were quantified using an ex vivo nerve-specific fluorescence assay on pig and human sciatic nerve. Results were used to perform quantitative structure-activity relationship (QSAR) modeling and to define the nerve-specific pharmacophore. All DSB analogs with positive ex vivo fluorescence were tested for in vivo nerve specificity in mice to assess the effect of biodistribution and clearance on nerve fluorescence signal. Two new DSB fluorophores with the highest nerve to muscle ratio were tested in pigs to confirm scalability.

High-throughput screening of small molecule ligands targeted to live bacteria surface.

The discovery of small molecule ligands targeted to the surface of live pathogenic bacteria would enable an entirely new class of antibiotics. We report the development and validation of a microarray-based high-throughput screening platform for bacteria that exploits 300 µm diameter chemical spots in a 1 in. × 3 in. nanolayered glass slide format. Using 24 model compounds and 4 different bacterial strains, we optimized the screening technology, including fluorophore-based optical deconvolution for automated scoring of affinity and cyan-magenta-yellow-key (CMYK) color-coding for scoring of both affinity and specificity. The latter provides a lossless, one-dimensional view of multidimensional data. By linking in silico analysis with cell binding affinity and specificity, we could also begin to identify the physicochemical factors that affect ligand performance. The technology we describe could form the foundation for developing new classes of antibiotics.

Rapid and Facile Microwave-Assisted Surface Chemistry for Functionalized Microarray Slides.

We describe a rapid and facile method for surface functionalization and ligand patterning of glass slides based on microwave-assisted synthesis and a microarraying robot. Our optimized reaction enables surface modification 42-times faster than conventional techniques and includes a carboxylated self-assembled monolayer, polyethylene glycol linkers of varying length, and stable amide bonds to small molecule, peptide, or protein ligands to be screened for binding to living cells. We also describe customized slide racks that permit functionalization of 100 slides at a time to produce a cost-efficient, highly reproducible batch process. Ligand spots can be positioned on the glass slides precisely using a microarraying robot, and spot size adjusted for any desired application. Using this system, we demonstrate live cell binding to a variety of ligands and optimize PEG linker length. Taken together, the technology we describe should enable high-throughput screening of disease-specific ligands that bind to living cells.

High-throughput small molecule identification using MALDI-TOF and a nanolayered substrate.

Encoderless combinatorial chemistry requires high-throughput product identification without the use of chemical or other tags. We developed a novel nanolayered substrate plate and combined it with a microarraying robot, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, and custom software to produce a high-throughput small molecule identification system. To optimize system performance, we spotted 5 different chemical entities, spanning a m/z range of 195 to 1338, in 20,304 spots for a total of 101,520 molecules. The initial spot identification rate was 99.85% (20,273 spots), and after a proofreading algorithm was added, 100% of 20,304 spots and 101,520 molecules were identified. An internal recalibration algorithm also significantly improved mass accuracy to as low as 45 ppm. Using this optimized system, 47 different chemical entities, spanning a m/z range of 138 to 1,592, were spotted over 5,076 spots and could be identified with 100% accuracy. Our study lays the foundation for improved encoderless combinatorial chemistry.

Nerve-highlighting fluorescent contrast agents for image-guided surgery.

Nerve damage is the major morbidity of many surgeries, resulting in chronic pain, loss of function, or both. The sparing of nerves during surgical procedures is a vexing problem because surrounding tissue often obscures them. To date, systemically administered nerve-highlighting contrast agents that can be used for nerve-sparing image-guided surgery have not been reported. In the current study, physicochemical and optical properties of 4,4'-[(2-methoxy-1,4-phenylene)di-(1E)-2,1-ethenediyl]bis-benzenamine (BMB) and a newly synthesized, red-shifted derivative 4-[(1E)-2-[4-[(1E)-2-[4-aminophenyl]ethenyl]-3-methoxyphenyl]ethenyl]-benzonitrile (GE3082) were characterized in vitro and in vivo. Both agents crossed the blood-nerve barrier and blood-brain barrier and rendered myelinated nerves fluorescent after a single systemic injection. Although both BMB and GE3082 also exhibited significant uptake in white adipose tissue, GE3082 underwent a hypsochromic shift in adipose tissue that provided a means to eliminate the unwanted signal using hyperspectral deconvolution. Dose and kinetic studies were performed in mice to determine the optimal dose and drug-imaging interval. The results were confirmed in rat and pig, with the latter used to demonstrate, for the first time, simultaneous fluorescence imaging of blood vessels and nerves during surgery using the FLARE™ (Fluorescence-Assisted Resection and Exploration) imaging system. These results lay the foundation for the development of ideal nerve-highlighting fluorophores for image-guided surgery.

Biodistribution and clearance of small molecule hapten chelates for pretargeted radioimmunotherapy.

PURPOSE: The favorable pharmacokinetics and clinical safety profile of metal-chelated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) suggests that it might be an ideal hapten for pretargeted radioimmunotherapy. In an effort to minimize hapten retention in normal tissues and determine the effect of various chemical adducts on in vivo properties, a series of DOTA-based derivatives were evaluated. PROCEDURES: Biodistribution and whole-body clearance were evaluated for (177)Lu-labeled DOTA, DOTA-biotin, a di-DOTA peptide, and DOTA-aminobenzene in normal CD1 mice. Kidney, liver, and bone marrow doses were estimated using standard Medical Internal Radiation Dose methodology. RESULTS: All haptens demonstrated similar low tissue and whole-body retention, with 2-4% of the injected dose remaining in mice 4 h postinjection. The kidney is predicted to be dose limiting for all (177)Lu-labeled haptens tested with an estimated kidney dose of approximately 0.1 mGy/MBq. CONCLUSIONS: We present here a group of DOTA-based haptens that exhibit rapid clearance and exceptionally low whole-body retention 4 h postinjection. Aminobenzene, tyrosine-lysine, and biotin groups have minimal effects on the blood clearance and biodistribution of (177)Lu-DOTA.

Molecular imaging agents specific for the annulus fibrosus of the intervertebral disk.

Low back pain is a prevalent medical condition that is difficult to diagnose and treat. Current imaging methods are unable to correlate pain reliably with spinal structures, and surgical removal of painful damaged or degenerating disks is technically challenging. A contrast agent specific for the intervertebral disk could assist in the detection, diagnosis, and surgical treatment of low back pain. The styryl pyridinium (FM) fluorophores were characterized and structure-activity relationships between chemical structure and in vivo uptake were established. Two novel FM fluorophores with improved optical properties for imaging the intervertebral disks were synthesized and evaluated in mice, rats, and pigs. After a single systemic injection, eight of eight FM fluorophores provided high-contrast imaging of the trigeminal ganglia, whereas six of eight provided high-contrast imaging of the dorsal root ganglia. Unexpectedly, three of eight FM fluorophores provided high-contrast imaging of annulus fibrosus tissue of the intervertebral disks, confirmed histologically. We present the first known contrast agent specific for the intervertebral disks and identify the chemical structural motif that mediates uptake. FM fluorophores could be used for image-guided surgery to assist in the removal of intervertebral disk and lay the foundation for derivatives for magnetic resonance imaging and positron emission tomography.

Context-independent, temperature-dependent helical propensities for amino acid residues.

Assigned from data sets measured in water at 2, 25, and 60 degrees C containing (13)C=O NMR chemical shifts and [theta](222) ellipticities, helical propensities are reported for the 20 genetically coded amino acids, as well as for norvaline and norleucine. These have been introduced by chemical synthesis at central sites within length-optimized, spaced, solubilized Ala(19) hosts. The resulting polyalanine-derived, quantitative propensity sets express for each residue its temperature-dependent but context-independent tendency to forego a coil state and join a preexisting helical conformation. At 2 degrees C their rank ordering is: P << G < H < C, T, N < S < Y, F, W < V, D < K < Q < I < R, M < L < E < A; at 60 degrees C the rank becomes: H, P < G < C < R, K < T, Y, F < N, V < S < Q < W, D < I, M < E < A < L. The DeltaDeltaG values, kcal/mol, relative to alanine, for the cluster T, N, S, Y, F, W, V, D, Q, imply that at 2 degrees C all are strong breakers: DeltaDeltaG(mean) = +0.63 +/- 0.11, but at 60 degrees C their breaking tendencies are dramatically attenuated and converge toward the mean: DeltaDeltaG(mean) = +0.25 +/- 0.07. Accurate modeling of helix-rich proteins found in thermophiles, mesophiles, and organisms that flourish near 0 degrees C thus requires appropriately matched propensity sets. Comparisons are offered between the temperature-dependent propensity assignments of this study and those previously assigned by the Scheraga group; the special problems that attend propensity assignments for charged residues are illustrated by lysine guest data; and comparisons of errors in helicity assignments from shifts and ellipticity data show that the former provide superior precision and accuracy.

Helix-coil energetics for helix formers and breakers reflect context and temperature: mutants of helically robust, guest-sensitive homopeptide hosts.

The natural amino acids are primarily helix breakers at the low assignment temperatures characteristic of many studies, but recent genomic analyses of thermophilic proteins suggest that at high temperatures, some breakers may become strong helix formers. Moreover, the breaker/former inventory has not been previously characterized at the physiologically relevant temperature of 37 degrees C. The versatility of 13C==O NMR chemical shifts as helicity reporters allows construction of two mutant peptide series, tailored to expand the range of temperature assignments for helical propensities and derived from the core hosts tL-Ala9XxxAla9-tL and tL-AlaNva4XxxNva4Ala9-tL, Nva=norvaline. For three limiting guests Xxx, the helix former Nva and the breakers Gly and Pro, we report wXxx[T] assignments at seven temperatures from 2 to 80 degrees C, validating our reasoning and paving the way for assignment of a definitive wXxx[T] data-base.