Jesus E. Gonzalez

Improved indicators of cell membrane potential that use fluorescence resonance energy transfer

BACKGROUNDnFluorescence detection of cell membrane potentials is an important technique in neurobiology, cell physiology and pharmaceutical screening, but traditional one-fluorophore indicators either respond too slowly or have limited sensitivity. Recently, we introduced two-component sensors based on the transfer of fluorescence resonance energy from fluorescent lectins bound on one side of the plasma membrane to highly fluorescent oxonol acceptors that electrophorese from one face of the membrane to the other in response to membrane potential.nnnRESULTSnWe have found that fluorescent lectins can often be advantageously replaced in such sensors by fluorescently labeled phospholipids. A coumarinlabeled phosphatidylethanolamine donor and a bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol acceptor gave the largest sensitivity of fluorescence ratio (>50% per 100 mV) ever reported. The response was also speeded several-fold by lengthening the mobile dye to the pentamethineoxonol analog, the <0.4 ms time constant of which was shorter than action potential durations. Photodynamic damage due to singlet oxygen was reduced by administering a natural carotenoid, astaxanthin.nnnCONCLUSIONSnVoltage-sensitive fluorescence resonance energy transfer already gives record-setting performance on single cells and will continue to be rationally improvable.

Cell-based assays and instrumentation for screening ion-channel targets

Ion channels are an important class of drug targets. They comprise the molecular basis for essential physiological functions including fluid secretion, electrolyte balance, bioenergetics and membrane excitability. High-throughput screening for ion-channel function requires sensitive, simple assays and instrumentation that will report ion channel activity in living cells. This article will review relevant assay technologies for ion channels and describe voltage-sensitive probes and instruments based on fluorescence resonance energy transfer (FRET) that enable ion-channel drug discovery.

Identification of Neural Circuits by Imaging Coherent Electrical Activity with FRET-Based Dyes

We show that neurons that underlie rhythmic patterns of electrical output may be identified by optical imaging and frequency-domain analysis. Our contrast agent is a two-component dye system in which changes in membrane potential modulate the relative emission between a pair of fluorophores. We demonstrate our methods with the circuit responsible for fictive swimming in the isolated leech nerve cord. The output of a motor neuron provides a reference signal for the phase-sensitive detection of changes in fluorescence from individual neurons in a ganglion. We identify known and possibly novel neurons that participate in the swim rhythm and determine their phases within a cycle. A variant of this approach is used to identify the postsynaptic followers of intracellularly stimulated neurons.

Intraoperative Tumor Detection Using a Ratiometric Activatable Fluorescent Peptide: A First-in-Human Phase 1 Study

BackgroundPositive surgical margins remain a significant challenge in breast cancer surgery. This report describes the use of a novel, first-in-human ratiometric activatable cell-penetrating peptide in breast cancer surgery.MethodsA two-part, multi-institutional phase 1 trial of AVB-620 with a 3+3 dose escalation and dose-expansion cohorts was conducted. The patients received an infusion of AVB-620 2–20xa0h before planned lumpectomy/mastectomy and sentinel node biopsy/axillary dissection. Imaging analysis was performed on images obtained from the surgical field as well as post-excision surgical specimens. Pathology reports were obtained to correlate imaging results with histopathologic data. Information on physical adverse events and laboratory abnormalities were recorded.ResultsA total of 27 patients received infusion of AVB-620 and underwent surgical excision of breast cancer. The findings showed no adverse events or laboratory values attributable to infusion of AVB-620. The 8-mg dose was selected from the dose-escalation cohort for use with the expansion cohort based on imaging data. Region-of-interest (ROI) imaging analysis from the 8-mg cohort demonstrated measurable changes between pathology confirmed tumor-positive and tumor-negative tissue.ConclusionIntraoperative imaging of surgical specimens after infusion with AVB-620 allowed for real-time tumor detection. Infusion of AVB-620 is safe and may improve intraoperative detection of malignant tissue during breast cancer operations.

Sensitive in vivo Visualization of Breast Cancer Using Ratiometric Protease-activatable Fluorescent Imaging Agent, AVB-620

With the goal of improving intraoperative cancer visualization, we have developed AVB-620, a novel intravenously administered, in vivo fluorescent peptide dye conjugate that highlights malignant tissue and is optimized for human use. Matrix metalloproteinases (MMPs) hydrolyze AVB-620 triggering tissue retention and a ratiometric fluorescence color change which is visualized using camera systems capable of imaging fluorescence and white light simultaneously. AVB-620 imaging visualizes primary tumors and demonstrated high in vivo diagnostic sensitivity and specificity (both >95%) for identifying breast cancer metastases to lymph nodes in two immunocompetent syngeneic mouse models. It is well tolerated and single-dose toxicology studies in rats determined a no-observed-adverse-effect-level (NOAEL) at >110-fold above the imaging and estimated human dose. Protease specificity and hydrolysis kinetics were characterized and compared using recombinant MMPs. To understand the human translation potential, an in vitro diagnostic study was conducted to evaluate the ability of AVB-620 to differentiate human breast cancer tumor from healthy adjacent tissue. Patient tumor tissue and healthy adjacent breast tissue were homogenized, incubated with AVB-620, and fluorogenic responses were compared. Tumor tissue had 2-3 fold faster hydrolysis than matched healthy breast tissue; generating an assay sensitivity of 96% and specificity of 88%. AVB-620 has excellent sensitivity and specificity for identifying breast cancer in mouse and human tissue. Significant changes were made in the design of AVB-620 relative to previous ratiometric protease-activated agents. AVB-620 has pharmaceutical properties, fluorescence ratio dynamic range, usable diagnostic time window, a scalable synthesis, and a safety profile that have enabled it to advance into clinical evaluation in breast cancer patients.

Monitoring integrated activity of individual neurons using FRET-based voltage-sensitive dyes

Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cells membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.