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Ronald T Borchardt

Publications and source records attributed to Ronald T Borchardt.

At least 19 recordsLinked to original sources
PubMed2006

Effects of ligand binding and oxidation on hinge-bending motions in S-adenosyl-L-homocysteine hydrolase.

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.

Adenosylhomocysteinase↗
PubMed2006

Effect of N-1 and N-2 residues on peptide deamidation rate in solution and solid state.

The deamidation kinetics of 7 model peptides (VYPNGA, VYGNGA, VFGNGA, VIGNGA, VGGNGA, VGPNGA, and VGYNGA) were studied at 70 degrees C in pH 10 buffer solutions and at 70 degrees C and 50% relative humidity in lyophilized solid formulations containing polyvinyl pyrrolidone (PVP). The disappearance of the model peptides from solution and solid-state formulations followed apparent first-order kinetics, proceeding to completion in solution. In the solid state, the reactions showed plateaus with approximately 10% to 30% of the model peptides remaining; this was thought to be due to reversible complexation of the peptides and the PVP followed by slow dissociation of the complexes. The residues immediately N-terminal to asparagine (N-1, N-2) influenced the rate of deamidation significantly in the solid state but had minimal effect in solution. Increases in the volume and hydrophobicity of the N-1 and N-2 residues decreased the rate of deamidation in the solid state, but neither parameter alone adequately accounted for the observed effects. An empirical model using a linear combination of volume and hydrophobicity was developed; it showed that the influences of the volume and the hydrophobicity of the residues in the N-1 and N-2 positions are approximately equally important for the N-1 and N-2 residues.

Amides↗
PubMed2006

Effects of amino acid chirality and the chemical linker on the cell permeation characteristics of cyclic prodrugs of opioid peptides.

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine whether the chirality of the amino acid asymmetric centers could influence the solution structure of the cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast, the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of the prodrugs.

Alanine↗
PubMed2006

Enzymes involved in the bioconversion of ester-based prodrugs.

Enzymes are essential for the activation of many prodrugs. In this review, the most important enzymes (e.g., paraoxonase, carboxylesterase, acetylcholinesterase, cholinesterase) involved in the bioconversion of ester-based prodrugs will be discussed in terms of their biology and biochemistry. Most of these enzymes fall into the category of hydrolytic enzymes. However, nonhydrolytic enzymes, including cytochrome P450s, can also catalyze the bioconversion of ester prodrugs and thus will be discussed here. Other factors influencing the ability of these enzymes to catalyze the bioconversion of ester-based prodrugs, particularly species and interindividual differences and stereochemical and structural features of the prodrugs, will be discussed.

Animals↗
PubMed2006

Editorial.

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Education, Pharmacy↗
PubMed2006

Editorial.

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Editorial↗
PubMed2006

Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization.

The asparagine (Asn) deamidation and aspartate (Asp) isomerization reactions are nonenzymatic intra-molecular reactions occurring in peptides and proteins that are a source of major stability concern in the formulation of these biomolecules. The mechanisms for the deamidation and isomerization reactions are similar since they both proceed through an intra-molecular cyclic imide (Asu) intermediate. The formation of the Asu intermediate, which involves the attack by nitrogen of the peptide backbone on the carbonyl carbon of the Asn or the Asp side chain, is the rate-limiting step in both the deamidation and the isomerization reactions at physiological pH. In this article, the influence of factors such as formulation conditions, protein primary sequence, and protein structure on the reactivity of Asn and Asp residues in proteins are reviewed. The importance of formulation conditions such as pH and solvent dielectric in influencing deamidation and isomerization reaction rates is addressed. Formulation strategies that could improve the stability of proteins to deamidation and isomerization reactions are described. The review is intended to provide information to formulation scientists, based on protein sequence and structure, to predict potential degradative sites on a protein molecule and to enable formulation scientists to set appropriate formulation conditions to minimize reactivity of Asn and Asp residues in protein therapeutics.

Amides↗
PubMed2005

Are L-adenosine and its derivatives substrates for S-adenosyl-L-homocysteine hydrolase?

Moffatt oxidation of 2',3'-O-isopropylidene-L-adenosine and treatment of the resulting crude 5'-aldehyde with hydroxylamine followed by deprotection gave L-adenosine 5'-carboxaldehyde oximes, whose enantiomers are known to be potent inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. The L-adenosine and its 5'-aldehyde oxime derivatives were found to be inactive as inhibitors of AdoHcy hydrolase. Docking calculations showed that binding of L-adenosine to AdoHcy hydrolase is weaker (higher energy) and less specific (larger number of clusters) compared to D-Ado.

Adenosine↗
PubMed2005

Domain motions and the open-to-closed conformational transition of an enzyme: a normal mode analysis of S-adenosyl-L-homocysteine hydrolase.

The structure and fluctuations of the enzyme S-adenosyl-L-homocysteine hydrolase (SAHH) are analyzed in an effort to explain its biological function. Besides the previously identified open structure, characteristic of the substrate-free enzyme, we find two distinct structures in enzyme-inhibitor complexes, the closed and closed-twisted conformers. Both closed conformers differ from the open form by a hinge bending motion of two large domains within each subunit, which isolate the inhibitor bound in the active site from the bulk solvent. The closed-twisted form further differs from the closed form by a rigid body twist of the two-subunit dimers. The local structural fluctuations of SAHH are analyzed by performing block normal mode analysis of the tetrameric enzyme in its three forms. For the open form, we find that the four lowest-frequency normal modes, corresponding to the collective motions of the protein with the largest amplitudes, are essentially combinations of the hinge bending deformations of the individual subunits. Thus, the mechanical properties of the open structure of SAHH lead to the presence of structural fluctuations in the direction of the open-to-closed conformational transition. A candidate for such a motion has been observed in previous fluorescence depolarization studies of the enzyme. Both structural and normal mode analyses indicate that residues 180-190 and 350-356 form hinge regions, connecting large domains which tend to move as rigid bodies in response to interactions with substrate, intermediates, and the product of the enzymatic reactions. We propose that these hinge regions play a crucial role in the enzymatic mechanism of SAHH. In contrast to the open form, normal mode calculations for the closed conformations show strong coupling of the hinge bending motions of the individual subunits to each other and to other low-frequency vibrations. Thus, information about structural changes related to reaction progress in one active site may be mechanically transmitted to other subunits of the protein, explaining the cooperativity found in the enzyme kinetics.

Adenine↗
PubMed2005

Effects of acidic N + 1 residues on asparagine deamidation rates in solution and in the solid state.

The deamidation kinetics of four model peptides (AcGQNGG, AcGQNDG, AcGQNEG, and AcGQNQG) were studied in solution (70 degrees C, pH 5-10) and in lyophilized solids [70 degrees C, 50% relative humidity, "effective pH" ('pH') 5-10] containing polyvinyl pyrrolidone. AcGQNGG, AcGQNEG, and AcGQNQG degraded exclusively through Asn deamidation, whereas AcGQNDG also displayed Asp isomerization, and Asp-Gly peptide bond cleavage. The pH/'pH'-rate profiles were consistent with a shift in the rate-determining step of Asn deamidation from carbonyl addition to expulsion of ammonia with increasing pH. In solution, AcGQNGG deamidated up to 38-fold faster than the other peptides, indicating the importance of steric effects of the N + 1 residue. AcGQNGG and AcGQNQG had up to 60 times slower rates of deamidation in the solid state than in solution. In contrast, the deamidation rates of AcGQNEG and AcGQNDG in the solid state were similar to those in solution. N + 1 Glu or Asp residue may enhance local hydration, so that the deamidation of Asn in the solid formulations actually proceeds in a solution-like environment.

Amides↗
PubMed2005

Effects of sucrose and mannitol on asparagine deamidation rates of model peptides in solution and in the solid state.

Asparagine (Asn) degradation kinetics in two model peptides, Gly-Gln-Asn-Gly-Gly (GQNGG) and Val-Tyr-Pro-Asn-Gly-Ala (VYPNGA), were studied at 50 degrees C in pH 7 buffer solutions in the presence and absence of 5% (w/v) sucrose or mannitol and at 50 degrees C and 30% relative humidity in solid samples lyophilized from these solutions. Solid formulations were characterized using Karl Fischer coulometric titration, thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), Fourier-transform infrared spectrometry (FTIR), and solid-state nuclear magnetic resonance (NMR) spectroscopy. GQNGG and VYPNGA showed similar pseudo first-order deamidation rates in solution in the absence of sucrose and mannitol. Adding 5% sucrose or mannitol decreased the rates by no more than 17%. The model peptides degraded 2- to 80-fold more slowly in the solid formulations of sucrose and mannitol than in 5% solutions of these carbohydrates. Ratios of deamidation rates of the model peptides depended upon the solid matrix. In the mannitol solid, the ratio of deamidation rates of GQNGG and VYPNGA (GQNGG:VYPNGA) was approximately 8, while in the sucrose solid, the model peptides deamidated at similar rates (GQNGG:VYPNGA congruent with 1). DSC showed the mannitol formulations to be largely amorphous immediately after lyophilization with some ordered, crystalline-like structure; the extent of ordered structure increased during storage as shown by FTIR and ssNMR. In contrast, the sucrose formulation was largely amorphous after lyophilization and remained so during storage. Together, the results showed that 5% sucrose or mannitol in solution does not significantly change the rates of Asn deamidation of the model peptides, while sucrose stabilizes the model peptides against deamidation more than mannitol in the solid state.

Asparagine↗
PubMed2005

Stability of oxymethyl-modified coumarinic acid cyclic prodrugs of diastereomeric opioid peptides in biological media from various animal species including human.

In vitro stability studies of oxymethyl-modified coumarinic acid (OMCA) cyclic prodrugs of the diastereomeric opioid peptides DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), [Ala2,D-Leu5]-Enk (H-Tyr-Ala-Gly-Phe-D-Leu-OH), [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH), and [Ala2,Leu5]-Enk (H-Tyr-Ala-Gly-Phe-Leu-OH) were conducted to evaluate how the chirality of specific amino acid residues (Ala2 and Leu5) in the peptide portion affects their bioconversion by esterases. The stability studies were conducted at 37 degrees C in plasma and tissue homogenates (liver and brain) from five animal species (rat, mouse, canine, guinea pig, and hamster) and human in an attempt to identify an animal species that had a "prodrug bioconversion profile" comparable to that of humans. Initially, the total esterase activity in these biological media was measured using p-nitrophenyl butyrate (PNPB) as a substrate. By repeating this activity assay in the presence of paraoxon, a potent esterase B inhibitor, it was possible to estimate the relative amounts of esterases B and esterases A/C in a biological sample. Stability studies of the cyclic prodrugs were carried out under identical conditions, that is, in the presence and absence of paraoxon. Significant differences in the rates of hydrolysis of the cyclic prodrugs were observed, particularly between cyclic prodrugs with differences in the chirality of the amino acid on the C-terminus of the peptide portion, for example, L-amino acids at the C-terminus hydrolyzed more rapidly than D-amino acids. This stereoselective hydrolysis was independent of the animal species but tended to be more pronounced in brain and liver homogenates compared to plasma. Increased esterase specific activity, as measured by PNPB, in the biological media did not necessarily correlate with increased bioconversion rates of the cyclic prodrugs. The enzymatic stability profiles of the cyclic prodrugs in biological media from canine and guinea pig most closely resembled the profiles from human biological media. Therefore, canine and guinea pig appear to be the most relevant animal models for conducting pharmacokinetic studies on these cyclic prodrugs of opioid peptides.

Alanine↗
PubMed2005

Deamidation of model beta-turn cyclic peptides in the solid state.

To investigate the importance of secondary structure on peptide deamidation in the solid state, two cyclic beta-turn peptides and their linear analogs were used as models of Asn residues in structured and unstructured domains, and incorporated into poly(vinyl pyrrolidone) (PVP)-based lyophilized solids. The secondary structure of the model peptides was determined in solution and the solid state using a combination of nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. The model beta-turn cyclic peptides were found to be type II beta-turns while the linear analogs were determined to be predominantly unstructured. Quantitatively, the cyclic peptides consisted of approximately 80% beta-turn while the linear analogs contained only 30%-35% beta-turn. To characterize the solid environment, T(g), and moisture content of the solid-state formulations were determined. Accelerated stability studies were conducted in the solid state at 37 degrees C using formulations lyophilized from solutions at pH 8.8 (0.1 M borate buffer). The effect of matrix mobility on solid-state deamidation was investigated by altering the moisture content through variation of relative humidity or the addition of a plasticizer. Cyclic peptides degraded 1.2-8 times slower than the linear analogs under all of the conditions studied. The observed rate constants, however, for all of the peptides decreased dramatically (four orders of magnitude) in the glassy solids. This suggests the greater importance of matrix mobility in solid-state degradation. Molecular dynamics (MD) simulations were also performed to explore the low energy, preferred state of the peptides, and determine the structure around the beta-turn.

Amides↗
PubMed2005

Significant differences in the disposition of cyclic prodrugs of opioid peptides in rats and guinea pigs following IV administration.

The stabilities of DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), the capped derivative Ac-DADLE-NH2, and the oxymethyl-coumarinic acid (OMCA)-based cyclic prodrug of DADLE and [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH) were determined at 37 degrees C in rat and guinea pig liver microsomes in the presence and absence of paraoxon, an esterase B inhibitor, and ketoconazole, a CYP3A4 inhibitor. These studies showed that the order of stability in microsomes was: DADLE >> Ac-DADLE-NH2 > OMCA-DADLE = OMCA-[D-Ala2,Leu5]-Enk. While paraoxon produced no significant effect on the stability of the studied compounds in liver microsomes, ketoconazole inhibited the metabolism, suggesting that the capped peptide and the cyclic prodrugs are substrates for cytochrome P450 enzymes. For pharmacokinetic studies, the cyclic prodrugs of DADLE and [D-Ala2,Leu5]-Enk were administered i.v. to rats and guinea pigs. Various biological fluids and tissue (brain, bile, and blood) were collected and analyzed for the free peptide and the prodrugs by high performance liquid chromatography with tandem mass spectrometric detection (LC-MS-MS). These studies showed that the conversion of the cyclic prodrugs to the respective linear peptides (i.e., DADLE and [D-Ala2,Leu5]-Enk) was rapid in rat and guinea pig. In terms of drug elimination, only trace amounts of OMCA-DADLE and OMCA-[D-Ala2,Leu5]-Enk were recovered in guinea pig bile (3.3% and 0.82%, respectively), while significant amounts were recovered in rat bile (38.1% and 51.7%, respectively). Brain uptake of the cyclic prodrugs in guinea pigs compared to previously determined brain uptake of OMCA-DADLE in rats was also significantly different. For OMCA-DADLE, the brain levels of the cyclic prodrug and DADLE in guinea pigs were approximately 80 and 8.5 times greater, respectively, than the levels observed in rat brain. The brain-to-plasma prodrug concentration ratios in guinea pigs (>or= 0.6) were significantly higher than the ratio observed in rats (0.01). These species differences are most likely due to the different substrate specificities of the efflux transporters that facilitate liver clearance of these prodrugs and limit their permeation into the brain.

Animals↗
PubMed2004

Stereoselective synthesis of sugar-modified enyne analogues of adenosine and uridine. Interaction with S-adenosyl-L-homocysteine hydrolase and antiviral and cytotoxic effects.

Sonogashira coupling of (E)-6'-iodohomovinyl nucleosides 1 with (trimethylsilyl)acetylene gave the conjugated 8'-(trimethylsilyl)enyne derivatives of the adenosine 2a and uridine 2b with expected E-stereochemistry. Desilylation of 2a,b with tetrabutylammonium fluoride followed by treatment with N-iodosuccinimide/AgNO(3) afforded 8'-iodoenynes 4a,b. Analogous coupling of (Z)-6'-iodohomovinyl nucleosides 7a,b produced (Z)-8'-(trimethylsilyl)enynes 8a,b, which were deprotected with aqueous trifluoroacetic acid to give the Z-enynes 9a,b. Stereoselective coupling of the adenosine 4'-acetylenic 11 and ethyl (Z)-3-bromoacrylate followed by deprotection gave the conjugated enyne system attached in the reverse orientation at C4' 13. Because of their diverse stereochemical attributes, deprotected enyne analogues 5a, 6a, 9a, and 13 derived from adenosine require a different vicinity for binding with S-adenosyl-l-homocysteine (AdoHcy) hydrolase and/or addition of enzyme-bound water across the conjugated enyne system. Enyne 5a and 8'-iodoenyne 6a produced time-dependent and concentration-dependent inhibition of AdoHcy hydrolase (K(i), 0.55 and 118.5 microM, respectively). No reduction in NAD(+) content of the enzyme and no iodide ion released were observed upon incubation of 6a with the enzyme, while incubation of 5a produced 30% reduction in the NAD(+) content of the enzyme. No specific antiviral activity was noted for 5a,b, 6a,b, 9a,b, and 13 against any of the viruses tested; the E-iodoenynes 6a and 6b inhibited HIV-1 virus (IC(50), 1.1 and 1.8 microM; selectivity index, 7 and 3, respectively). The E-enyne 5a showed activity against cytomegalovirus at a concentration (EC(50), 30 microM) that was 3- to 10-fold lower than the cytotoxic concentration.

Adenosine↗
PubMed2004

Phe-Gly dipeptidomimetics designed for the di-/tripeptide transporters PEPT1 and PEPT2: synthesis and biological investigations.

A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester 1, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (2a), (R)- and (S)-hydroxyethylidene (3a and 4a), and (R)- and (S)-hydroxyethylene (5a and 6a) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere 2a and the natural substrate Phe-Gly (K(i) values of 18.8 and 14.6 microM, respectively). Also the affinities for hPEPT1 are in the same range (K(i) values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C(4)-position for this class of compounds. Furthermore, the intracellular uptake of 2a-4a in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of 2a in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of 3a and 4a was observed. Transepithelial transport studies also indicated active transport of 2a across Caco-2 monolayers.

Animals↗
PubMed2004

Bidirectional transport of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein, across MDCK-MDR1 cell monolayers.

The bidirectional permeation characteristics of rhodamine 123 and Hoechst 33342, fluorescence probes of the binding sites on P-glycoprotein (P-gp), across monolayers of MDCK cells transfected with the human MDR1 gene (MDCK-MDR1) were investigated. The ratios of the apparent permeability coefficients (P(app)) of rhodamine 123 and Hoechst 33342 flux measured in the basolateral (BL) to apical (AP) direction versus the flux in the AP-to-BL direction (P(app BL-to-AP)/P(app AP-to-BL)) were 115 and 177, respectively. The P-gp inhibitor GF-120918 could significantly reduce the polarized efflux of both rhodamine 123 and Hoechst 33342. Rhodamine 123 appeared to "stimulate" the polarized efflux of Hoechst 33342 across MDCK-MDR1 cell monolayers. In contrast, Hoechst 33342 partially inhibited the polarized efflux of rhodamine 123 across these cell monolayers whereas daunorubicin partially inhibited the polarized efflux of both rhodamine 123 and Hoechst 33342. The uptake characteristics of rhodamine 123 and Hoechst 33342 in MDCK-MDR1 cells were measured in the absence and presence of GF-120918 and known P-gp substrates (Hoechst 33342, rhodamine 123, and daunorubicin). The uptake of rhodamine 123 and Hoechst 33342 in MDCK-MDR1 cells was enhanced more than twofold by inclusion of GF-120918 (2 microM) in the incubation medium. Daunorubicin (160 microM) increased the relative fluorescence unit (RFU) values of cytoplasm-associated rhodamine 123 by up to 30%. However, daunorubicin (40 microM) and rhodamine 123 (5 microM) decreased the RFU values of cell membrane-associated Hoechst 33342 by 70% and 40%, respectively. To further explore what appears to be a "stimulatory" effect of daunorubicin and rhodamine 123 on the uptake of Hoechst 33342 and a stimulatory effect of daunorubicin on Hoechst 33342 transport across cell monolayer, uptake of Hoechst 33342 into liposomes in the presence and absence of GF-120918, daunorubicin, and rhodamine 123 was determined. GF-120918 exhibited no effect on the RFU values of liposome-associated Hoechst 33342. In contrast, rhodamine 123 and daunorubicin decreased the fluorescence of liposome-associated Hoechst 33342 suggesting these molecules were either quenching the fluorescence of this chemical probe or displacing it from the lipid bilayer. In conclusion, these bidirectional transport data indicate that rhodamine 123 and Hoechst 33342 are excellent substrates of P-gp in MDCK-MDR1 cells. The ability of Hoechst 33342 to partially inhibit the polarized efflux of rhodamine 123 is consistent with these substrates binding to the same site on P-gp. In contrast, the ability of rhodamine 123 to apparently "stimulate" the efflux of Hoechst 33342 in both the transport and uptake experiments suggests the substrates might bind to different sites on P-gp. However, experimental results using liposomes suggested that this "stimulation" phenomenon by rhodamine 123 on Hoechst 33342 uptake and efflux might simply be an artifact. Thus, the use of Hoechst 33342 to probe the binding sites on a membrane-bound protein such as P-gp might be problematic.

ATP Binding Cassette Transporter, Subfamily B, Mem↗