The Large-neutral Amino Acid Transporter 1 (LAT-1)-a sodiumindependent exchanger of amino acids, thyroid hormones, and prescription drugs-is highly expressed in the blood-brain barrier and various types of cancer. LAT-1 plays an important role in cancer development as well as in mediating drug and nutrient delivery across the blood-brain barrier, making it a key drug target. Here, we identify four LAT-1 ligands, including one chemically novel substrate, by comparative modeling, virtual screening, and experimental validation. These results may rationalize the enhanced brain permeability of two drugs, including the anticancer agent acivicin. Finally, two of our hits inhibited proliferation of a cancer cell line by distinct mechanisms, providing useful chemical tools to characterize the role of LAT-1 in cancer metabolism.arge-neutral Amino Acid Transporter 1 (LAT-1) is a sodiumindependent exchanger found in the brain, testis, and placenta, where it mediates transport of large-neutral amino acids (e.g., tyrosine) and thyroid hormones (e.g., triiodothyronine) across the cell membrane (1). More specifically, LAT-1 is highly expressed in the blood-and brain-facing membranes of the blood-brain barrier (BBB) to supply the central nervous system (CNS) with essential nutrients and to help maintain the neural microenvironment (2). LAT-1 is also an important drug target because it transports several prescription drugs, such as the antiparkinsonian drug L-dopa and the anticonvulsant gabapentin, across the BBB, thereby enabling their pharmacologic effects (3,4). This function at the BBB has made LAT-1 a target for drug delivery by modifying CNS-impermeable drugs such that they become LAT-1 substrates and have enhanced BBB penetration (5, 6).In addition, LAT-1 expression levels are increased in many types of cancer, including non-small-cell lung cancer and glioblastoma multiforme (GBM) (7,8). LAT-1 expression increases as cancers progress, leading to higher expression levels in highgrade tumors and metastases (9). In particular, LAT-1 plays a key role in cancer-associated reprogrammed metabolic networks by supplying growing tumor cells with essential amino acids that are used as nutrients to build biomass and signaling molecules to enhance proliferation by activating progrowth pathways such as the mammalian target of rapamycin (mTOR) pathway (10). Furthermore, inhibiting LAT-1 function reduces tumor cell proliferation, indicating that it may be a viable target for novel anticancer therapies (11-13). A cancer drug targeting LAT-1 can therefore be a LAT-1 inhibitor that deprives the cancer cells of nutrients or a cytotoxic LAT-1 substrate with an intracellular target (e.g., a metabolic enzyme).LAT-1 is a large protein with 12 putative membrane-spanning helices (14). To transport solutes across the membrane, LAT-1 binds SLC3A2, a glycoprotein with a single membrane-spanning helix that serves as a chaperone for LAT-1 (14). The atomic structure of human LAT-1 is not known, but LAT-1 exhibits significant sequence similarity to prokar...

The goal of this study was to determine the role of an influx copper transporter, CTR1, in the ototoxicity induced by cisplatin, a potent anticancer platinum analog used in the treatment of a variety of solid tumors. As determined through reverse transcriptase-PCR (RT-PCR), quantitative RT-PCR, Western blot, and immunohistochemistry, mouse CTR1 (Ctr1) was found to be abundantly expressed and highly localized at the primary sites of cisplatin toxicity in the inner ear, mainly outer hair cells (OHCs), inner hair cells, stria vascularis, spiral ganglia, and surrounding nerves in the mouse cochlea. A CTR1 substrate, copper sulfate, decreased the uptake and cytotoxicity of cisplatin in HEI-OC1, a cell line that expresses many molecular markers reminiscent of OHCs. Small interfering RNA-mediated knockdown of Ctr1 in this cell line caused a corresponding decrease in cisplatin uptake. In mice, intratympanic administration of copper sulfate 30 min before intraperitoneal administration of cisplatin was found to prevent hearing loss at click stimulus and 8, 16, and 32 kHz frequencies. To date, the utility of cisplatin remains severely limited because of its ototoxic effects. The studies described in this report suggest that cisplatin-induced ototoxicity and cochlear uptake can be modulated by administration of a CTR1 inhibitor, copper sulfate. The possibility of local administration of CTR1 inhibitors during cisplatin therapy as a means of otoprotection is thereby raised.

The norepinephrine transporter (NET) transports norepinephrine from the synapse into presynaptic neurons, where norepinephrine regulates signaling pathways associated with cardiovascular effects and behavioral traits via binding to various receptors (e.g., β2-adrenergic receptor). NET is a known target for a variety of prescription drugs, including antidepressants and psychostimulants, and may mediate off-target effects of other prescription drugs. Here, we identify prescription drugs that bind NET, using virtual ligand screening followed by experimental validation of predicted ligands. We began by constructing a comparative structural model of NET based on its alignment to the atomic structure of a prokaryotic NET homolog, the leucine transporter LeuT. The modeled binding site was validated by confirming that known NET ligands can be docked favorably compared to nonbinding molecules. We then computationally screened 6,436 drugs from the Kyoto Encyclopedia of Genes and Genomes (KEGG DRUG) against the NET model. Ten of the 18 high-scoring drugs tested experimentally were found to be NET inhibitors; five of these were chemically novel ligands of NET. These results may rationalize the efficacy of several sympathetic (tuaminoheptane) and antidepressant (tranylcypromine) drugs, as well as side effects of diabetes (phenformin) and Alzheimer's (talsaclidine) drugs. The observations highlight the utility of virtual screening against a comparative model, even when the target shares less than 30% sequence identity with its template structure and no known ligands in the primary binding site.

High-density lipoprotein (HDL) mediates reverse cholesterol transport (RCT), wherein excess cholesterol is conveyed from peripheral tissues to the liver and steroidogenic organs. During this process HDL continually transitions between subclass sizes, each with unique biological activities. For instance, RCT is initiated by the interaction of lipid-free/lipid-poor apolipoprotein A-I (apoA-I) with ABCA1, a membrane-associated lipid transporter, to form nascent HDL. Because nearly all circulating apoA-I is lipid-bound, the source of lipid-free/lipid-poor apoA-I is unclear. Lecithin:cholesterol acyltransferase (LCAT) then drives the conversion of nascent HDL to spherical HDL by catalyzing cholesterol esterification, an essential step in RCT. To investigate the relationship between HDL particle size and events critical to RCT such as LCAT activation and lipid-free apoA-I production for ABCA1 interaction, we reconstituted five subclasses of HDL particles (rHDL of 7.8, 8.4, 9.6, 12.2, and 17.0 nm in diameter, respectively) using various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, free cholesterol, and apoA-I. Kinetic analyses of this comprehensive array of rHDL particles suggest that apoA-I stoichiometry in rHDL is a critical factor governing LCAT activation. Electron microscopy revealed specific morphological differences in the HDL subclasses that may affect functionality. Furthermore, stability measurements demonstrated that the previously uncharacterized 8.4 nm rHDL particles rapidly convert to 7.8 nm particles, concomitant with the dissociation of lipid-free/lipid-poor apoA-I. Thus, lipid-free/lipid-poor apoA-I generated by the remodeling of HDL may be an essential intermediate in RCT and HDL's in vivo maturation.