Alec Tate Harootunian

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.

Practical design criteria for a dynamic ratio imaging system

Problems encountered and solutions devised during the construction of a productive Ca2(+)-imaging system are discussed. Many of these relate to the rapid and interactive nature of experiments on cytosolic Ca2+. The emphasis on accurate photometric quantitation of dynamically changing images contrasts with that of most image-processing software packages, which concentrate either on leisurely massage of static images or on descriptions of lateral motions of objects without concern for their brightnesses. Particularly important goals for Ca2(+)-imaging include real-time ratioing, psychophysically effective display formats, ease of experiment annotation, mass storage of image sequences, automated extraction of time courses and population statistics, aids to presenting images for seminars and publications, and program modifiability.

Cytosolic Ca2+ oscillations in REF52 fibroblasts: Ca2+-stimulated IP3 production or voltage-dependent Ca2+ channels as key positive feedback elements

Oscillations in cytosolic free calcium concentrations ([Ca2+]i) can be elicited in REF52 fibroblasts by three different modes of stimulation. We have previously demonstrated that [Ca2+]i oscillations result when these cells are simultaneously depolarized and stimulated with a hormone linked to phosphoinositide breakdown. Further evidence is now presented that such oscillations are linked to fluctuations in the concentration of IP3 and the Ca2+ content of an IP3-sensitive Ca2+ store. [Ca2+]i oscillations can also be generated in REF52 cells either by direct stimulation of G-proteins with GTP gamma S or AlF4- or by destabilizing the membrane potential and opening voltage-dependent calcium channels. This report compares the different types of oscillations and their mechanisms.

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.

Imaging and manipulation of cytosolic ions and messengers during cell activation

Summary Optical methods have recently become available for continuously imaging the free concentrations of important ions and second messengers such as calcium, sodium and hydrogen inside living cells. These ion levels are found to undergo remarkable changes upon stimulation of quiescent cells with growth factors known to stimulate phosphoinositide breakdown. In serum-starved REF-52 fibroblasts, growth factors such as serum, vasopressin, or PDGF (platelet-derived growth factor) cause intracellular [Na+] to increase from about 4 mm to 8 mm. If mitogen treatment is combined with pharmacological depolarization of the membrane potential, repetitive [Ca2+]j spikes result in these rat fibroblasts. The mechanism of this oscillation has been investigated by light-flash release of intracellular messengers such as inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), Ca2+, and diacylglycerol, as well as more traditional biochemical techniques. The key feedback pathway appears to be Ca2+-stimulation of phospholipase C production of Ins(1,4,5)P3.