Proteasome Inhibitors Bortezomib and Carfilzomib Stimulate the Transport Activity of Human Organic Anion Transporter 1.

Organic anion transporter 1 (OAT1), expressed at the basolateral membrane of renal proximal tubule epithelial cells, mediates the renal excretion of many clinically important drugs. Previous study in our laboratory demonstrated that ubiquitin conjugation to OAT1 leads to OAT1 internalization from the cell surface and subsequent degradation. The current study showed that the ubiquitinated OAT1 accumulated in the presence of the proteasomal inhibitors MG132 and ALLN rather than the lysosomal inhibitors leupeptin and pepstatin A, suggesting that ubiquitinated OAT1 degrades through proteasomes. Anticancer drugs bortezomib and carfilzomib target the ubiquitin-proteasome pathway. We therefore investigate the roles of bortezomib and carfilzomib in reversing the ubiquitination-induced downregulation of OAT1 expression and transport activity. We showed that bortezomib and carfilzomib extremely increased the ubiquitinated OAT1, which correlated well with an enhanced OAT1-mediated transport of p-aminohippuric acid and an enhanced OAT1 surface expression. The augmented OAT1 expression and transport activity after the treatment with bortezomib and carfilzomib resulted from a reduced rate of OAT1 degradation. Consistent with this, we found decreased 20S proteasomal activity in cells that were exposed to bortezomib and carfilzomib. In conclusion, this study identified the pathway in which ubiquitinated OAT1 degrades and unveiled a novel role of anticancer drugs bortezomib and carfilzomib in their regulation of OAT1 expression and transport activity. SIGNIFICANCE STATEMENT: Bortezomib and carfilzomib are two Food and Drug Administration-approved anticancer drugs, and proteasome is the drug target. In this study, we unveiled a new role of bortezomib and carfilzomib in enhancing OAT1 expression and transport activity by preventing the degradation of ubiquitinated OAT1 in proteasomes. This finding provides a new strategy in regulating OAT1 function that can be used to accelerate the clearance of drugs, metabolites, or toxins and reverse the decreased expression under disease conditions.

Solution-processed, top-emitting, microcavity polymer light-emitting diodes for the pure red, green, blue and near white emission.

Top-emitting microcavity polymer light-emitting diodes (TMPLEDs) are of great significance for active matrix PLED displays with high color purity. However, the complex device structures of highly efficient microcavity organic light-emitting diodes fabricated by the full vapor deposition technology are not suitable for solution-processed PLEDs. Solution-processed TMPLEDs with simple device structures are promising candidates for large-area, mass production display techniques. In this work, three strategies were used to apply microcavity into PLEDs: (1) double Ag electrodes performed as the mirrors of cavity, instead of a multi-layer Bragg reflector, which simplified the device structure and fabrication process; (2) three solution-processed functional layers were specially designed for avoiding the inter-infiltration between the different solutions and to improve the interface contacts; (3) high order microcavities were utilized according to the optical simulation results, in which thick EMLs benefited from thickness control and reproductivity. As a result, the full-color emission including pure red, green, blue was realized, and quasi-white light was also achieved from a single polymer emitting material. The achievement of color purity always requires the sacrifice of part of the current efficiency due to the spectra narrowing, while the higher current efficiency of green TMPLED (10.08 cd A-1) compared to that of non-cavity PLED (~8.60 cd A-1) cast a light on future improvements.

AG490, a JAK2-specific inhibitor, downregulates the expression and activity of organic anion transporter-3.

Human organic anion transporter-3 (hOAT3) is richly expressed in the kidney, where it plays critical roles in the secretion of clinically important drugs, including anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. In the current study, we examined the role of AG490, a specific inhibitor of the Janus tyrosine kinase 2 (JAK2), in hOAT3 transport activity in the kidney COS-7 cells. AG490 induced a time- and concentration-dependent inhibition of hOAT3-mediated uptake of estrone sulfate, a prototypical substrate for the transporter. The inhibitory effect of AG490 correlated with a reduced expression of hOAT3 at the cell surface. Our lab previously demonstrated that Nedd4-2, a ubiquitin ligase, down regulates OAT expression and transport activity by enhancing OAT ubiquitination, which leads to an internalization of OAT from cell surface to intracellular compartments and subsequent degradation. In the current study, we showed that treatment of hOAT3-expressing cells with AG490 resulted in an enhanced hOAT3 ubiquitination and degradation, which was accompanied by a strengthened association of Nedd4-2 with hOAT3 and a reduction in Nedd4-2 phosphorylation. SiRNA knockdown of endogenous Nedd4-2 abrogated the effects of AG490 on hOAT3. In summary, our study demonstrated that AG490 regulates hOAT3 expression and transport activity through the modulation of Nedd4-2.

The activity of organic anion transporter-3: Role of dexamethasone.

Human organic anion transporter-3 (hOAT3) is richly expressed in the kidney, where it plays critical roles in the secretion, from the blood to urine, of clinically important drugs, such as anti-viral therapeutics, anti-cancer drugs, antibiotics, antihypertensives, and anti-inflammatories. In the current study, we examined the role of dexamethasone in hOAT3 transport activity in the kidney HEK293 cells. Cis-inhibition study showed that dexamethasone exhibited a concentration-dependent inhibition of hOAT3-mediated uptake of estrone sulfate, a prototypical substrate for the transporter, with IC50 value of 49.91 µM. Dixon plot analysis revealed that inhibition by dexamethasone was competitive with a Ki = 47.08 µM. In contrast to the cis-inhibition effect of dexamethasone, prolonged incubation (6 h) of hOAT3-expressing cells with dexamethasone resulted in an upregulation of hOAT3 expression and transport activity, kinetically revealed as an increase in the maximum transport velocity Vmax without meaningful alteration in substrate-binding affinity Km. Such upregulation was abrogated by GSK650394, a specific inhibitor for serum- and glucocorticoid-inducible kinases (sgk). Dexamethasone also enhanced sgk1 phosphorylation. Our study demonstrated that dexamethasone exhibits dual effects on hOAT3: it is a competitive inhibitor for hOAT3-mediated transport, and interestingly, when entering the cells, it stimulates hOAT3 expression and transport activity through sgk1.

PKC/Nedd4-2 Signaling Pathway Regulates the Cell Surface Expression of Drug Transporter hOAT1.

Human organic anion transporter-1 (hOAT1) regulates the absorption, distribution, and excretion of a wide range of clinically important drugs. Our previous work demonstrated that hOAT1 is a dynamic membrane transporter, constitutively internalizing from and recycling back to the cell plasma membrane. Short-term activation (<30 minutes) of protein kinase C (PKC) promotes the attachment of a lysine 48-linked polyubiquitin chain to hOAT1, a process catalyzed by ubiquitin ligase neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2). The ubiquitination of hOAT1 then triggers an accelerated endocytosis of the transporter from plasma membrane, which results in reduced hOAT1 expression at the cell surface and decreased hOAT1 transport activity. In the present study, we investigated the long-term effect of PKC on hOAT1. We showed that long-term activation (>2 hours) of PKC significantly enhanced hOAT1 degradation, and such action was partially blocked by ubiquitin mutant Ub-K48R, which has its lysine (K) 48 mutated to arginine (R) and is incapable of forming a K48-linked polyubiquitin chain. The ubiquitin ligase Nedd4-2 was also found to augment hOAT1 degradation. These results suggest that PKC-regulated and Nedd4-2-catalyzed attachment of a lysine 48-linked polyubiquitin chain to hOAT1 is important for hOAT1 stability. We further showed through coimmunoprecipitation experiments that there was a direct association between hOAT1 and Nedd4-2, and such interaction was weakened when the WW3 and WW4 domains of the ligase were mutated. Mutating WW3 and WW4 domains of the ligase also impaired its ability to ubiquitinate hOAT1. Therefore, WW3 and WW4 domains of Nedd4-2 are critical for its association with and modulation of the transporter.

SGK1/Nedd4-2 signaling pathway regulates the activity of human organic anion transporters 3.

Human organic anion transporter 3 (hOAT3) is localized at the basolateral membrane of renal proximal tubule cells and facilitates the renal secretion of numerous clinical drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, antihypertension drugs and anti-inflammatories. The present study explored the role of serum and glucocorticoid-inducible kinase 1 (sgk1) in the regulation of hOAT3. It was shown that over-expression of sgk1 in hOAT3-expressing cells stimulated hOAT3 transport activity by enhancing the transporter expression at the plasma membrane, kinetically reflected as an increased maximal transport velocity Vmax without substantial change in the substrate-binding affinity Km . In contrast, treatment of cells with the sgk-specific inhibitor GSK650394 resulted in a dose-dependent inhibition of hOAT3 transport activity. Evidence was further provided that sgk1 regulation of hOAT3 activity was mediated by ubiquitin ligase Nedd4-2, an enzyme previously shown to have an inhibitory effect on hOAT3. It was shown that sgk1 phosphorylated Nedd4-2, weakened the association between Nedd4-2 and hOAT3, and decreased hOAT3 ubiquitination. Functionally, the sgk1-stimulated hOAT3 transport activity was attenuated in the presence of a ligase-dead mutant of Nedd4-2. In summary, the investigation established for the first time that sgk1 stimulates hOAT3 transport activity by interfering with the inhibitory effect of Nedd4-2 on the transporter.

Posttranslational Regulation of Organic Anion Transporters by Ubiquitination: Known and Novel.

Organic anion transporters (OATs) encoded by solute carrier 22 family are localized in the epithelia of multiple organs, where they mediate the absorption, distribution, and excretion of a diverse array of negatively charged environmental toxins and clinically important drugs. Alterations in the expression and function of OATs play important roles in intra- and interindividual variability of the therapeutic efficacy and the toxicity of many drugs. As a result, the activity of OATs must be under tight regulation so as to carry out their normal functions. The regulation of OAT transport activity in response to various stimuli can occur at several levels such as transcription, translation, and posttranslational modification. Posttranslational regulation is of particular interest, because it usually happens within a very short period of time (minutes to hours) when the body has to deal with rapidly changing amounts of substances as a consequence of variable intake of drugs, fluids, or meals as well as metabolic activity. This review article highlights the recent advances from our laboratory in uncovering several posttranslational mechanisms underlying OAT regulation. These advances offer the promise of identifying targets for novel strategies that will maximize therapeutic efficacy in drug development.

Nedd4-2 but not Nedd4-1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1.

Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.

The role of Nedd4-1 WW domains in binding and regulating human organic anion transporter 1.

Human organic anion transporter 1 (hOAT1), expressed at the basolateral membrane of kidney proximal tubule cells, mediates the active renal secretion of a diverse array of clinically important drugs, including anti-human immunodeficiency virus therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We have previously demonstrated that posttranslational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The present study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of neural precursor cell expressed, developmentally downregulated (Nedd)4-1, an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific small interfering RNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and rat kidney slices and immunofluorescence experiments in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains (Mut-WW1: V210W/H212G, Mut-WW2: V367W/H369G, Mut-WW3: I440W/H442G, and Mut-WW4: I492W/H494G, respectively), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild-type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.

An Essential Role of Nedd4-2 in the Ubiquitination, Expression, and Function of Organic Anion Transporter-3.

Organic anion transporter-3 (OAT3) is a member of the organic anion transporter family that mediates the body disposition of a diverse array of clinically important drugs. We previously demonstrated that activation of protein kinase C (PKC) inhibits OAT3 transport activity by accelerating OAT3 internalization from cell surface into intracellular compartments. In the current study, we established that PKC-induced inhibition of OAT3 transport activity occurred through an enhanced OAT3 ubiquitination, a process catalyzed by an E3 ubiquitin-protein ligase Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2). Overexpression of Nedd4-2 enhanced OAT3 ubiquitination, decreased OAT3 expression at the cell surface, and inhibited OAT3 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-2/C821A or siRNA knockdown of endogenous Nedd4-2 had opposite effects on OAT3. Furthermore, immunoprecipitation experiments conducted both in culture cells and with rat kidney slices showed that there was a physical interaction between OAT3 and Nedd4-2. In conclusion, our results provided the first evidence that Nedd4-2 is an important regulator for OAT3 ubiquitination, expression, and transport activity.

Preface to Special Issue: Drug Transporters: Regulation and Roles in Therapeutic Strategies.

Drug transporters are membrane proteins, mediating, across cell membranes, the absorption, distribution, and excretion of a diverse array of endogenous and exogenous substances such as nutrients, metabolites, toxins, and drugs [...].

Topotecan and Ginkgolic Acid Inhibit the Expression and Transport Activity of Human Organic Anion Transporter 3 by Suppressing SUMOylation of the Transporter.

Organic anion transporter 3 (OAT3), expressed at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous metabolites, environmental toxins, and clinically important drugs. An earlier investigation from our laboratory revealed that OAT3 expression and transport activity can be upregulated by SUMOylation, a post-translational modification that covalently conjugates SUMO molecules to substrate proteins. Topotecan is a semi-synthetic derivative of the herbal extract camptothecin, approved by the FDA to treat several types of cancer. Ginkgolic acid (GA) is one of the major components in the extract of Ginkgo biloba leaves that has long been used in food supplements for preventing dementia, high blood pressure, and supporting stroke recovery. Both topotecan and GA have been shown to affect protein SUMOylation. In the current study, we tested our hypothesis that topotecan and GA may regulate OAT3 SUMOylation, expression, and transport function. Our data show that the treatment of OAT3-expressing cells with topotecan or GA significantly decreases the SUMOylation of OAT3 by 50% and 75%, respectively. The same treatment also led to substantial reductions in OAT3 expression and the OAT3-mediated transport of estrone sulfate, a prototypical substrate. Such reductions in cell surface expression of OAT3 correlated well with an increased rate of OAT3 degradation. Mechanistically, we discovered that topotecan enhanced the association between OAT3 and the SUMO-specific protease SENP2, a deSUMOylation enzyme, which contributed to the significant decrease in OAT3 SUMOylation. In conclusion, this study unveiled a novel role of topotecan and GA in inhibiting OAT3 expression and transport activity and accelerating OAT3 degradation by suppressing OAT3 SUMOylation. During comorbidity therapies, the use of topotecan or Ginkgo biloba extract could potentially decrease the transport activity of OAT3 in the kidneys, which will in turn affect the therapeutic efficacy and toxicity of many other drugs that are substrates for the transporter.

Three ubiquitination sites of organic anion transporter-1 synergistically mediate protein kinase C-dependent endocytosis of the transporter.

Organic anion transporter-1 (OAT1) mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. Therefore, understanding the regulation of OAT1 has profound clinical significance. We previously established that OAT1 constitutively internalizes from and recycles back to cell surface and that activation of protein kinase C (PKC) inhibits OAT1 activity by promoting ubiquitination of the transporter, which then leads to an accelerated internalization of the transporter from cell surface to intracellular compartments. In the current study, we showed that PKC isoform PKCα was responsible for OAT1 ubiquitination. To directly address the role of OAT1 ubiquitination, we then generated two OAT1 mutants, each having multiple lysines (K) simultaneously mutated to arginine (R). One mutant K163/297/303/315/321R lost sensitivities to PKC-induced inhibition of transport activity, to PKC-induced ubiquitination, and to PKC-induced acceleration of transporter internalization. Further dissecting each lysine in this mutant, we identified Lys297, Lys303, and Lys315 as being the ubiquitin conjugation sites. Of interest, mutating any one of the three lysines prevented the ubiquitin conjugation to the other two lysines, suggesting that Lys297, Lys303, and Lys315 may form an optimal structure to interact with ubiquitination machineries. This is the first demonstration that Lys297, Lys303, and Lys315 play a synergistic role in PKC-regulated OAT1 ubiquitination, trafficking, and transport activity.

Lysine 48-linked polyubiquitination of organic anion transporter-1 is essential for its protein kinase C-regulated endocytosis.

Organic anion transporter-1 (OAT1) mediates the body's disposition of a diverse array of environmental toxins and clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. We had previously established that OAT1 undergoes constitutive internalization from and recycling back to the cell surface and that acute activation of protein kinase C (PKC) inhibits OAT1 activity by reducing OAT1 cell-surface expression through accelerating its internalization from cell surface to intracellular compartments. However, the underlying mechanisms are poorly understood. In the current study, we provide novel evidence that acute activation of PKC significantly enhances OAT1 ubiquitination both in vitro and ex vivo. We further show that ubiquitination of cell-surface OAT1 increases in cells transfected with dominant negative mutant of dynamin-2, a maneuver blocking OAT1 internalization, which suggests that OAT1 ubiquitination proceeds before OAT1 internalization. Mass spectroscopy has revealed that ubiquitination of OAT1 consists of polyubiquitin chains, primarily through lysine 48 linkage. Transfection of cells with the dominant negative mutant of ubiquitin Ub-K48R, which prevents the formation of Lys48-linked polyubiquitin chains, abolishes PKC-stimulated OAT1 ubiquitination and internalization. Together, our findings demonstrate for the first time that Lys48-linked polyubiquitination is essential for PKC-regulated OAT1 trafficking.

Chloroquine and Hydroxychloroquine, as Proteasome Inhibitors, Upregulate the Expression and Activity of Organic Anion Transporter 3.

Organic anion transporter 3 (OAT3), at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous widely used drugs. Earlier investigation from our laboratory revealed that ubiquitin conjugation to OAT3 leads to OAT3 internalization from the cell surface, followed by degradation in the proteasome. In the current study, we examined the roles of chloroquine (CQ) and hydroxychloroquine (HCQ), two well-known anti-malarial drugs, in their action as proteasome inhibitors and their effects on OAT3 ubiquitination, expression, and function. We showed that in cells treated with CQ and HCQ, the ubiquitinated OAT3 was considerably enhanced, which correlated well with a decrease in 20S proteasome activity. Furthermore, in CQ- and HCQ-treated cells, OAT3 expression and OAT3-mediated transport of estrone sulfate, a prototypical substrate, were significantly increased. Such increases in OAT3 expression and transport activity were accompanied by an increase in the maximum transport velocity and a decrease in the degradation rate of the transporter. In conclusion, this study unveiled a novel role of CQ and HCQ in enhancing OAT3 expression and transport activity by preventing the degradation of ubiquitinated OAT3 in proteasomes.

The regulation of human organic anion transporter 4 by insulin-like growth factor 1 and protein kinase B signaling.

Human organic anion transporter 4 (hOAT4), mainly expressed in the kidney and placenta, is essential for the disposition of numerous drugs, toxins, and endogenous substances. Insulin-like growth factor 1 (IGF-1) is a hormone generated in the liver and plays important roles in systemic growth, development, and metabolism. In the current study, we explored the regulatory effects of IGF-1 and downstream signaling on the transport activity, protein expression, and SUMOylation of hOAT4. We showed that IGF-1 significantly increased the transport activity, expression, and maximal transport velocity Vmax of hOAT4 in kidney-derived cells. This stimulatory effect of IGF-1 on hOAT4 activity was also confirmed in cells derived from the human placenta. The increased activity and expression were correlated well with the reduced degradation rate of hOAT4 at the cell surface. Furthermore, IGF-1 significantly increased hOAT4 SUMOylation, and protein kinase B (PKB)-specific inhibitors blocked the IGF-1-induced regulations on hOAT4. In conclusion, our study demonstrates that the hepatic hormone IGF-1 regulates hOAT4 expressed in the kidney and placenta through the PKB signaling pathway. Our results support the remote sensing and signaling theory, where OATs play a central role in the remote communications among distal tissues.

Pancreatic Hormone Insulin Modulates Organic Anion Transporter 1 in the Kidney: Regulation via Remote Sensing and Signaling Network.

Organic anion transporter 1 (OAT1) expressed in the kidney plays an important role in the elimination of numerous anionic drugs used in the clinic. We report here that insulin, a pancreas-secreted hormone, regulated the expression and activity of kidney-specific OAT1 both in cultured cells and in rats. We showed that treatment of OAT1-expressing cells with insulin led to an increase in OAT1 expression, transport activity, and SUMOylation. Such insulin-induced increase was blocked by afuresertib, a specific inhibitor for protein kinase B (PKB), suggesting insulin regulates OAT1 through PKB signaling pathway. Furthermore, insulin stimulated transport activity and SUMOylation of endogenously expressed OAT1 in rat kidneys. In conclusion, our data support a remote sensing and signaling model, in which OAT1 plays an essential role in intercellular and inter-organ communication and in maintaining local and whole-body homeostasis. Such complex and dedicated communication is carried out by insulin, and PKB signaling and membrane sorting.

Inhibition of proteasome, but not lysosome, upregulates organic anion transporter 3 in vitro and in vivo.

Organic anion transporter 3 (OAT3), an indispensable basolateral membrane transporter predominantly distributed in the kidney proximal tubules, mediated the systemic clearance of substrates including clinical drugs, nutrients, endogenous and exogenous metabolites, toxins, and critically sustains body homeostasis. Preliminary data in this study showed that classical proteasome inhibitors (e.g., MG132), but not lysosome inhibitors, significantly increased the OAT3 ubiquitination and OAT3-mediated transport of estrone sulfate (ES) in OAT3 stable expressing cells, indicating that proteasome rather than lysosome is involved in the intracellular fate of OAT3. Next, bortezomib and carfilzomib, two FDA-approved and widely applied anticancer agents through selective targeting proteasome, were further used to define the role of inhibiting proteasome in OAT3 regulation and related molecular mechanisms. The results showed that 20S proteasome activity in cell lysates was suppressed with bortezomib and carfilzomib treatment, leading to the increased OAT3 ubiquitination, stimulated transport activity of ES, enhanced OAT3 surface and total expression. The upregulated OAT3 function by proteasome inhibition was attributed to the augment in maximum transport velocity and stability of membrane OAT3. Lastly, in vivo study using Sprague Dawley rats validated that proteasome inhibition using bortezomib induced enhancement of OAT3 ubiquitination and membrane expression in kidney. These data suggest that activity of proteasome but not lysosome could have an impact on the physiological function of OAT3, and proteasome displayed a promising target for OAT3 regulation in vitro and in vivo, and could be used in restoring OAT3 impairment under pathological conditions, avoiding OAT3-associated toxicity and diseases, ensuring drug efficacy and safety.

Potent inhibitors of human organic anion transporters 1 and 3 from clinical drug libraries: discovery and molecular characterization.

Transporter-mediated drug-drug interactions in the kidney dramatically influence the pharmacokinetics and other clinical effects of drugs. Human organic anion transporters 1 (hOAT1) and 3 (hOAT3) are the major transporters in the basolateral membrane of kidney proximal tubules, mediating the rate-limiting step in the elimination of a broad spectrum of drugs. In the present study, we screened two clinical drug libraries against hOAT1 and hOAT3. Of the 727 compounds screened, 92 compounds inhibited hOAT1 and 262 compounds inhibited hOAT3. When prioritized based on the peak unbound plasma concentrations of these compounds, three inhibitors for hOAT1 and seven inhibitors for hOAT3 were subsequently identified with high inhibitory potency (>95%). Computational analyses revealed that inhibitors and noninhibitors can be differentiated from each other on the basis of several physicochemical features, including number of hydrogen-bond donors, number of rotatable bonds, and topological polar surface area (TPSA) for hOAT1; and molecular weight, number of hydrogen-bond donors and acceptors, TPSA, partition coefficient (log P(7.4)), and polarizability for hOAT3. Pharmacophore modeling identified two common structural features associated with inhibitors for hOAT1 and hOAT3, viz., an anionic hydrogen-bond acceptor atom, and an aromatic center separated by ∼5.7 Å. Such model provides mechanistic insights for predicting new OAT inhibitors.