This white paper provides updated International Transporter Consortium (ITC) recommendations on transporters that are important in drug development following the 3 ITC workshop. New additions include prospective evaluation of organic cation transporter 1 (OCT1) and retrospective evaluation of organic anion transporting polypeptide (OATP)2B1 because of their important roles in drug absorption, disposition, and effects. For the first time, the ITC underscores the importance of transporters involved in drug-induced vitamin deficiency (THTR2) and those involved in the disposition of biomarkers of organ function (OAT2 and bile acid transporters).

Citation: Sreekumar PG, Chothe P, Sharma KK, et al. Antiapoptotic properties of a-crystallin-derived peptide chaperones and characterization of their uptake transporters in human RPE cells. Invest Ophthalmol Vis Sci. 2013;54:278754: -279854: . DOI:10.1167 PURPOSE. The chaperone proteins, a-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer a-crystallin-derived mini-chaperone peptides (a-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells. METHODS.Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer aA-or aB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, a-crystallin-derived mini-peptides and recombinant full-length aB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for a-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated aB-crystallin mini-chaperone peptides from H 2 O 2 -induced cell death was studied.RESULTS. Primary hfRPE cells exposed to oxidative stress and either aA-or aB-crystallin minichaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length aB-crystallin was minimal while a time-dependent uptake of aB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing aB-crystallin mini-chaperone were also taken up and protected hfRPE from H 2 O 2 -induced cell death at significantly lower concentrations than free aB-crystallin minichaperone peptide.CONCLUSIONS. aA-and aB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of aB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.Keywords: a-crystallin, chaperone peptides, oxidative stress, RPE protection, oligopeptide transporters T he superfamily of small heat shock proteins (sHSPs) has attracted considerable attention in recent years because of its multifunctional cellular properties. The human genome encodes 10 members of the sHSP family, among which aAcrystallin and aB-crystallin are considered important members. 1 Both aA-and aB-crystallins have been studied extensively in the lens for their chaperone and related functions. However, recent studies have identified several novel functions for aA-and aBcrystallins in retina and other tissues in addition to their wellrecognized chaperone function. 2 Both a-crystallins are expressed in RPE cells and in the retina; higher expression of aBcrystallin was found in the RPE while aA-crystallin was found mostly in photoreceptors and astroglial and Mül...

Ascorbic acid (Vitamin C) has a critical role in bone formation and osteoblast differentiation, but very little is known about the molecular mechanisms of ascorbic acid entry into bone marrow stromal cells (BMSCs). To address this gap in knowledge, we investigated the identity of the transport system that is responsible for the uptake of ascorbic acid into bone marrow stromal cells (BMSCs). First, we examined the expression of the two known isoforms of the sodium-coupled ascorbic acid transporter, namely SVCT1 and SVCT2, in BMSCs (Lin-ve Sca1+ve) and bone at the mRNA level. Only SVCT2 mRNA was detected in BMSCs and bone. Uptake of ascorbic acid in BMSCs was Na+-dependent and saturable. In order to define the role of SVCT2 in BMSC differentiation into osteoblasts, BMSCs were stimulated with osteogenic media for different time intervals, and the activity of SVCT2 was monitored by ascorbic acid uptake. SVCT2 expression was up-regulated during the osteogenic differentiation of BMSCs; the expression was maximal at the earliest phase of differentiation. Subsequently, osteogenesis was inhibited in BMSCs upon knock-down of SVCT2 by lentivirus shRNA. We also found that the expression of the SVCT2 could be negatively or positively modulated by the presence of oxidant (Sin-1) or antioxidant (Ascorbic acid) compounds, respectively, in BMSCs. Furthermore, we found that this transporter is also regulated with age in mouse bone. These data show that SVCT2 plays a vital role in the osteogenic differentiation of BMSCs and that its expression is altered under conditions associated with redox reaction. Our findings could be relevant to bone tissue engineering and bone related diseases such as osteoporosis in which oxidative stress and aging plays important role.

Breast cancer resistance protein (BCRP; ABCG2) is an efflux transporter that plays an important role in multidrug resistance to antineoplastic drugs. The identification of drugs as BCRP inhibitors could aid in designing better therapeutic strategies for cancer treatment and will be critical for identifying potential drug-drug interactions. In the present study, we applied ligand-based virtual screening combined with experimental testing for the identification of novel drugs that can possibly interact with BCRP. Bayesian and pharmacophore models generated with known BCRP inhibitors were validated with an external test set. The resulting models were applied to predict new potential drug candidates from a database with more than 2000 FDA-approved drugs. Thirty-three drugs were tested in vitro for their inhibitory effects on BCRP-mediated transport of [(3)H]-mitoxantrone in MCF-7/AdrVp cells. Nineteen drugs were identified with significant inhibitory effect on BCRP transport function. The combined strategy of computational and experimental approaches in this paper has suggested potential drug candidates and thus represents an effective tool for rational identification of modulators of other proteins.

ARPE-19 cells as well as primary mouse and human fetal RPE cells express the newly discovered oligopeptide transporter SOPT2, and the transporter is induced by HIV-1 Tat in ARPE-19 cells.