Modeling the transport and inhibition kinetics of the human multidrug resistance transporter P-glycoprotein in MDCK-II cells

The human multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of radiolabeled substrates and study inhibition using pair wise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single kinetically relevant efflux-connected binding site. We conclude that there are at least two kinetically distinct efflux pathways through P-gp and the binding sites connected to these pathways may not be exclusive forany drug. Binding of these substrates must be cooperative which can be either positive or negative based on the substrate-inhibitor pair. From the transport and inhibition of digoxin and loperamide, we found that other transporters are responsible for uptake of these drugs into the cells and for recycling them into and from the apical membrane once P-gp has pumped them out. Each drug has a particular IC-50 for inhibiting transport of another. Exhaustive computer simulations of drug transport in the presence of “virtual inhibitors” showed that the fitted IC50 values overestimate the intrinsic dissociation constant, KI,Aq and an equation showed that this overestimate is contributed by a convolution of cell and substrate parameters. The smaller the passive permeability of the substrate, the greater is the magnitude of the overestimate of the inhibitor’s dissociation constant by its IC50. These conclusions are valid for any membrane transporter whose substrates and inhibitors must pass a permeability barrier to reach their binding site on the transporter.Ph.D., Cellular and Molecular Biology -- Drexel University, 200

Fitting the Elementary Rate Constants of the P-gp Transporter Network in the hMDR1-MDCK Confluent Cell Monolayer Using a Particle Swarm Algorithm

P-glycoprotein, a human multidrug resistance transporter, has been extensively studied due to its importance to human health and disease. In order to understand transport kinetics via P-gp, confluent cell monolayers overexpressing P-gp are widely used. The purpose of this study is to obtain the mass action elementary rate constants for P-gp's transport and to functionally characterize members of P-gp's network, i.e., other transporters that transport P-gp substrates in hMDR1-MDCKII confluent cell monolayers and are essential to the net substrate flux. Transport of a range of concentrations of amprenavir, loperamide, quinidine and digoxin across the confluent monolayer of cells was measured in both directions, apical to basolateral and basolateral to apical. We developed a global optimization algorithm using the Particle Swarm method that can simultaneously fit all datasets to yield accurate and exhaustive fits of these elementary rate constants. The statistical sensitivity of the fitted values was determined by using 24 identical replicate fits, yielding simple averages and standard deviations for all of the kinetic parameters, including the efflux active P-gp surface density. Digoxin required additional basolateral and apical transporters, while loperamide required just a basolateral tranporter. The data were better fit by assuming bidirectional transporters, rather than active importers, suggesting that they are not MRP or active OATP transporters. The P-gp efflux rate constants for quinidine and digoxin were about 3-fold smaller than reported ATP hydrolysis rate constants from P-gp proteoliposomes. This suggests a roughly 3∶1 stoichiometry between ATP hydrolysis and P-gp transport for these two drugs. The fitted values of the elementary rate constants for these P-gp substrates support the hypotheses that the selective pressures on P-gp are to maintain a broad substrate range and to keep xenobiotics out of the cytosol, but not out of the apical membrane

The ABCs of finding a good antibody: How to find a good antibody, validate it, and publish meaningful data [version 1; referees: 2 approved, 1 approved with reservations]

Finding an antibody that works for a specific application can be a difficult task. Hundreds of vendors offer millions of antibodies, but the quality of these products and available validation information varies greatly. In addition, several studies have called into question the reliability of published data as the primary metric for assessing antibody quality. We briefly discuss the antibody quality problem and provide best practice guidelines for selecting and validating an antibody, as well as for publishing data generated using antibodies

Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational ShutoffS⃞

Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)2 (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-κB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 μM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 μM with marked suppression at >100 μM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2α (eIF2α) kinase. Indeed, we found a marked (≈4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2α-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2α kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2α kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes

Hepatic Heme-Regulated Inhibitor (HRI) Eukaryotic Initiation Factor 2α Kinase: A Protagonist of Heme-Mediated Translational Control of CYP2B Enzymes and a Modulator of Basal Endoplasmic Reticulum Stress Tone

We have reported previously that the hepatic heme-regulated inhibitor (HRI)-eukaryotic initiation factor 2α (eIF2α) kinase is activated in acute heme-deficient states, resulting in translational shut-off of global hepatic protein synthesis, including phenobarbital (PB)-mediated induction of CYP2B enzymes in rats. These findings revealed that heme regulates hepatic CYP2B synthesis at the translational level via HRI. As a proof of concept, we have now employed a genetic HRI-knockout (KO) mouse hepatocyte model. In HRI-KO hepatocytes, PB-mediated CYP2B protein induction is no longer regulated by hepatic heme availability and proceeds undeterred even after acute hepatic heme depletion. It is noteworthy that genetic ablation of HRI led to a small albeit significant elevation of basal hepatic endoplasmic reticulum (ER) stress as revealed by the activation of ER stress-inducible RNA-dependent protein kinase-like ER-integral (PERK) eIF2α-kinase, and induction of hepatic protein ubiquitination and ER chaperones Grp78 and Grp94. Such ER stress was further augmented after PB-mediated hepatic protein induction. These findings suggest that HRI normally modulates the basal hepatic ER stress tone. Furthermore, because HRI exists in both human and rat liver in its heme-sensitive form and is inducible by cytochrome P450 inducers such as PB, these findings are clinically relevant to acute heme-deficient states, such as the acute hepatic porphyrias. Activation of this exquisitely sensitive heme sensor would normally protect cells by safeguarding cellular energy and nutrients during acute heme deficiency. However, similar HRI activation in genetically predisposed persons could lead to global translational arrest of physiologically relevant enzymes and proteins, resulting in the severe and often fatal clinical symptoms of the acute hepatic porphyrias

Liver Cytochrome P450 3A Ubiquitination in Vivo by gp78/Autocrine Motility Factor Receptor and C Terminus of Hsp70-interacting Protein (CHIP) E3 Ubiquitin Ligases: PHYSIOLOGICAL AND PHARMACOLOGICAL RELEVANCE*

CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drug-drug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function

Liver Cytochrome P450 3A Endoplasmic Reticulum-associated Degradation: A MAJOR ROLE FOR THE p97 AAA ATPase IN CYTOCHROME P450 3A EXTRACTION INTO THE CYTOSOL*

The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function