Verapamil Binding Research Articles

Influence of Aromatic-Group Structure on Cardiotropic Activity of Alkoxyphenyltriazaalkanes

Previously, ALM-802 (N1-(2,3,4-trimethoxybenzyl)-N2-{2-[(2,3,4-trimethoxybenzyl)amino]ethyl}-1,2-ethanediamine trihydrochloride) and ALM-803 (N1-(3,4,5-trimethoxybenzyl)-N2-{2-[(3,4,5-trimethoxybenzyl) amino]ethyl}-1,2-ethanediamine trihydrochloride), which combined pharmacophores of the free fatty-acid oxidation inhibitors trimetazidine and ranolazine and the slow Ca2+-channel blocker verapamil, were synthesized by us. These compounds possessed anti-ischemic and antiarrhythmic activities. Herein, the new compound ALM-843 (N1-(3,4-dimethoxybenzyl)-N2-{2-[(3,4-dimethoxybenzyl)amino]ethyl}-1,2-ethanediamine trihydrochloride), which exhibited only antiarrhythmic activity, was prepared. The results led to the hypothesis that the antiarrhythmic activity of the linear alkoxyphenyltriazaalkanes was due to the presence of a 3,4-dimethoxyphenyl pharmacophore, which is also present in verapamil. This hypothesis was confirmed using molecular docking to show that the above alkoxyphenyltriazaalkanes bound similarly to the verapamil binding site of the voltage-gated Ca2+-channel.

Search for ABCB1 Modulators Among 2-Amine-5-Arylideneimidazolones as a New Perspective to Overcome Cancer Multidrug Resistance.

Multidrug resistance (MDR) is a severe problem in the treatment of cancer with overexpression of glycoprotein P (Pgp, ABCB1) as a reason for chemotherapy failure. A series of 14 novel 5-arylideneimidazolone derivatives containing the morpholine moiety, with respect to two different topologies (groups A and B), were designed and obtained in a three- or four-step synthesis, involving the Dimroth rearrangement. The new compounds were tested for their inhibition of the ABCB1 efflux pump in both sensitive (parental (PAR)) and ABCB1-overexpressing (MDR) T-lymphoma cancer cells in a rhodamine 123 accumulation assay. Their cytotoxic and antiproliferative effects were investigated by a thiazolyl blue tetrazolium bromide (MTT) assay. For active compounds, an insight into the mechanisms of action using either the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp was performed. The safety profile in vitro was examined. Structure–activity relationship (SAR) analysis was discussed. The most active compounds, representing both 2-substituted- (11) and Dimroth-rearranged 3-substituted (18) imidazolone topologies, displayed 1.38–1.46 fold stronger efflux pump inhibiting effects than reference verapamil and were significantly safer than doxorubicin in cell-based toxicity assays in the HEK-293 cell line. Results of mechanistic studies indicate that active imidazolones are substrates with increasing Pgp ATPase activity, and their dye-efflux inhibition via competitive action on the Pgp verapamil binding site was predicted in silico.

Identification of Verapamil Binding Sites Within Human Kv1.5 Channel Using Mutagenesis and Docking Simulation.

The phenylalkylamine class of L-type Ca2+ channel antagonist verapamil prolongs the effective refractory period (ERP) of human atrium, which appears to contribute to the efficacy of verapamil in preventing reentrant-based atrial arrhythmias including atrial fibrillation. This study was designed to investigate the molecular and electrophysiological mechanism underlying the action of verapamil on human Kv1.5 (hKv1.5) channel that determines action potential duration and ERP in human atrium. Site-directed mutagenesis created 10 single-point mutations within pore region of hKv1.5 channel. Wholecell patch-clamp method investigated the effect of verapamil on wild-type and mutant hKv1.5 channels heterologously expressed in Chinese hamster ovary cells. Docking simulation was conducted using open-state homology model of hKv1.5 channel pore. Verapamil preferentially blocked hKv1.5 channel in its open state with IC50 of 2.4±0.6 μM (n = 6). The blocking effect of verapamil was significantly attenuated in T479A, T480A, I502A, V505A, I508A, L510A, V512A and V516A mutants, compared with wild-type hKv1.5 channel. Computer docking simulation predicted that verapamil is positioned within central cavity of channel pore and has contact with Thr479, Thr480, Val505, Ile508, Ala509, Val512, Pro513 and Val516. Verapamil acts as an open-channel blocker of hKv1.5 channel, presumably due to direct binding to specific amino acids within pore region of hKv1.5 channel, such as Thr479, Thr480, Val505, Ile508, Val512 and Val516. This blocking effect of verapamil on hKv1.5 channel appears to contribute at least partly to prolongation of atrial ERP and resultant antiarrhythmic action on atrial fibrillation in humans.

Specific N-demethylation of verapamil by cytochrome P450 from Streptomyces griseus ATCC 13273.

Norverapamil, the N-demethylated derivative of verapamil, is a novel promising leading compound for attenuating multidrug resistance with less side effects compared with verapamil. However, the efficient synthetic method for norverapamil is absent. In this study, an innovative biotechnological method based on enzymatic catalysis was presented for the high-efficient production of norverapamil. CYP105D1, a cytochrome P450 from Streptomyces griseus ATCC 13273, was identified to carry out a one-step specific N-demethylation of verapamil along with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) as the redox partner. Docking calculations rationalized the specific N-demethylation observed in experiment and identified important amino acid residues for verapamil binding. Furthermore, a CYP105D1-based whole-cell system in E. coli BL21(DE3) was established and optimized for highly efficient N-demethylation of verapamil. The bioconversion rate of verapamil by the whole cell system came up to 60.16% within 24 hours under the optimized conditions. These results demonstrated the high potential of CYP105D1-based biocatalytic system for norverapamil production.

Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels

Background/Aims: The human-voltage gated K_v1.3 channel (hK_v1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hK_v1.3 channels. We characterized the effect of verapamil on currents through hK_v1.3 channels paying special attention to the on-rate (k_on) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated. Methods: Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hK_v1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate k_on of verapamil. Results: The on-rate of verapamil to block current through hK_v1.3_T419C mutant channels is similar to that obtained for hK_v1.3_wt channels whereas the on-rate of verapamil to block currents through hK_v1.3_L417C and hK_v1.3_L418C mutant channels was ∼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hK_v1.3_L346C and the double mutant hK_v1.3_L346C_L418C channel was ∼ 2 times slower compared to that obtained in the wt channel. The hK_v1.3_I420C mutant channel reduced the on-rate of verapamil to block currents ∼ 6 fold. Conclusions: We conclude that position 420 in hK_v1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hK_v1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hK_v1.3_L346C and hK_v1.3_L346C_L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current.

Design, Synthesis and Calcium Channel Blocking Activity of Diltiazem- Verapamil Hybrid Molecules

The current manuscript describes the design, synthesis, and in vitro testing of four thioacetanilides with diltiazemverapamil hybrid structural features as potential calcium channel blockers. The current hybrid strategy of drug design aimed to generate compounds that could span, with a single compound, the trans-membrane locales where the two drugs bind, with the ultimate goal of increasing the blocking activity. The latter, was assessed by measuring the inhibitory effects, expressed as IC50, on calcium-induced contractions of potassium depolarized isolated rat aorta strips. The assessment of the binding locales was determined by incubating the test compound with aortic strips for two different periods, 10-minutes and 2-hours, before adding the contractile calcium ions to the assay medium. Diltiazem IC50 values were 0.26 and 0.14 μM, after 10-minutes and 2-hours, respectively, reflecting less than two fold increase in activity and confirming previous reports that its locale of binding in mostly on the exterior side of the membrane. On the other hand, verapamil IC50 values were 0.47 and 0.14 μM after 10-minutes and 2-hour incubation respectively, reflecting approximately a 3-4 fold increase in activity and confirming previous reports that it binds mainly to the interior domains of the membrane. The four designed hybrid compounds showed, after 10-minute incubation, an IC50 value range of 3.7-12.0 μM, and after 2-hour incubation an IC50 range of 0.78-2.12 μM, reflecting approximately a 5-fold increase in activity suggesting more similarity to the verapamil binding profile. The data indicate that the designed compounds are with moderate activities, but generally less active as calcium channel blockers than either of the two parent drugs.

Blood–brain barrier P-glycoprotein function in Alzheimer's disease

A major pathological hallmark of Alzheimer's disease is accumulation of amyloid-β in senile plaques in the brain. Evidence is accumulating that decreased clearance of amyloid-β from the brain may lead to these elevated amyloid-β levels. One of the clearance pathways of amyloid-β is transport across the blood-brain barrier via efflux transporters. P-glycoprotein, an efflux pump highly expressed at the endothelial cells of the blood-brain barrier, has been shown to transport amyloid-β. P-glycoprotein function can be assessed in vivo using (R)-[(11)C]verapamil and positron emission tomography. The aim of this study was to assess blood-brain barrier P-glycoprotein function in patients with Alzheimer's disease compared with age-matched healthy controls using (R)-[(11)C]verapamil and positron emission tomography. In 13 patients with Alzheimer's disease (age 65 ± 7 years, Mini-Mental State Examination 23 ± 3), global (R)-[(11)C]verapamil binding potential values were increased significantly (P = 0.001) compared with 14 healthy controls (aged 62 ± 4 years, Mini-Mental State Examination 30 ± 1). Global (R)-[(11)C]verapamil binding potential values were 2.18 ± 0.25 for patients with Alzheimer's disease and 1.77 ± 0.41 for healthy controls. In patients with Alzheimer's disease, higher (R)-[(11)C]verapamil binding potential values were found for frontal, parietal, temporal and occipital cortices, and posterior and anterior cingulate. No significant differences between groups were found for medial temporal lobe and cerebellum. These data show altered kinetics of (R)-[(11)C]verapamil in Alzheimer's disease, similar to alterations seen in studies where P-glycoprotein is blocked by a pharmacological agent. As such, these data indicate that P-glycoprotein function is decreased in patients with Alzheimer's disease. This is the first direct evidence that the P-glycoprotein transporter at the blood-brain barrier is compromised in sporadic Alzheimer's disease and suggests that decreased P-glycoprotein function may be involved in the pathogenesis of Alzheimer's disease.

Influence of Lignocaine on Plasma Protein Binding and Pharmacokinetics of Verapamil in Dogs

The effect of lignocaine (lidocaine) on the plasma protein binding of verapamil has been studied in-vitro and in-vivo in dogs. The binding of verapamil was ca 85%. In-vitro addition of lignocaine at therapeutic concentrations displaced verapamil from its plasma binding sites. Lignocaine in this regard was equipotent with tris(2-butoxyethyl)phosphate, suggesting an interaction at the level of alpha 1-acid glycoprotein binding sites. On in-vivo administration of 4 mg kg-1 in a bolus to dogs in which steady state concentrations of verapamil were present, the free fraction of verapamil increased transiently. During the lignocaine maintenance infusion, it then decreased to a level higher than that before administration of the local anaesthetic. The free verapamil concentrations increased suddenly upon the administration of the lignocaine loading dose, and then returned to values slightly higher than those before lignocaine. After a bolus injection of verapamil during a lignocaine infusion, the verapamil total plasma concentrations were lower than during a saline infusion, but the free concentrations were not different. The volume of distribution of verapamil was increased, whereas the blood clearance had not changed; the lignocaine infusion did not change the hepatic blood flow, as measured by indocyanine green clearance. These results show that lignocaine displaces verapamil in-vitro and in-vivo from its plasma protein binding sites, but the ensuing pharmacokinetic changes do not lead to significant changes in free verapamil concentrations.

Physicochemical interactions between drugs and superdisintegrants

Abstract We have evaluated the interactions between superdisintegrants and drugs with different physicochemical characteristics, which may affect the in-vivo absorption e.g. after mucosal administration. The binding of sodium salicylate, naproxen, methyl hydroxybenzoate (methylparaben), ethyl hydroxybenzoate (ethylparaben), propyl hydroxybenzoate (propylparaben), atenolol, alprenolol, diphenhydramine, verapamil, amitriptyline and cetylpyridinium chloride monohydrate (CPC) to different superdisintegrants (sodium starch glycolate (SSG), croscarmellose sodium (CCS) and crospovidone) and one unsubstituted comparator (starch) was studied spectrophotometrically. An indication of the in-vivo effect was obtained by measuring the interactions at physiological salt concentrations. SSG was investigated more thoroughly to obtain release profiles and correlation between binding and ionic strength. The results showed that the main interactions with the anionic hydrogels formed by SSG and CCS were caused by ion exchange, whereas the neutral crospovidone exhibited lipophilic interactions with the non-ionic substances. The effect of increased ionic strength was most pronounced at low salt concentrations and the ion exchange interactions were almost completely eradicated at physiological conditions. The release profile of diphenhydramine was significantly affected by the addition of salt. It was thus concluded that the choice of buffer was of great importance for in-vitro experiments with ionic drugs. At physiological salt concentrations the interactions did not appear to be strong enough to influence the in-vivo bioavailability of any of the drug molecules.

Does a Single Mutation in the P-Loop Open a Novel Current Pathway Beside the Central A-Pore in the Human Voltage-Gated Potassium Channel Kv1.3

Voltage-gated potassium channels are membrane proteins containing a potassium-selective ion conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel can disclose another ion permeation pathway through the voltage sensing domain (S1-S4) beside the α-pore, the so called ω-pore described by Tombola et al. 2005 (Neuron45:379-388). We investigated a mutant voltage-gated hKv1.3 channel where the substitution of a cysteine in the pore-loop at position 388 (Shaker position 438) generated a current through the α-pore, and an inward-current at hyperpolarizing potentials carried by different cations (Li+∼Na+>NH4+>Cs+>K+∼Rb+). This observed inward current appeared similar to the ω-current and was not affected by the α-pore blocker CTX, in contrary to the currents through the α-pore of the hKv1.3_V388C mutant channel. Verapamil, which is acting from the intracellular side, could block both, the α-current with an IC50 of 3.5 μM and the observed inward-current at hyperpolarizing potentials with an IC50 of 2.3 μM in the hKv1.3_V388C mutant channel. Due to the block of inward-current by verapamil we suppose that the observed inward-current runs through the verapamil binding site in the cavity between S6-S6 of two adjacent subunits in the hKv1.3_V388C mutant channel. We hypothesize that the hKv1.3_V388C mutation generated a channel with a second ion conducting pathway distinct from the α-pore, but not identical to the ω-pathway, running through one part of the hKv1.3 verapamil binding site. The entry of the novel pathway is presumably located at the backside of Y395 (Shaker position 445), proceeds plane parallel to the α-pore, ending between S5 and S6 at the intracellular side of one α-subunit.Supported by grants from the 4SC AG (Martinsried) and the DFG (Gr848/14-1).

Studies of verapamil binding to human serum albumin by high-performance affinity chromatography

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high-performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g. the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 10 4 M −1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37 °C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/ S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e. the warfarin–azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 10 4 M −1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with l-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body.

State-Dependent Verapamil Block of the Cloned Human Cav3.1 T-Type Ca2+ Channel

Verapamil is a potent phenylalkylamine antihypertensive believed to exert its therapeutic effect primarily by blocking high-voltage-activated L-type calcium channels. It was the first clinically used calcium channel blocker and remains in clinical use, although it has been eclipsed by other calcium channel blockers because of its short half-life and interactions with other channels. In addition to blocking L-type channels, it has been reported to block T-type (low-voltage activated) calcium channels. This type of cross-reactivity is likely to be beneficial in the effective control of blood pressure. Although the interactions of T channels with a number of drugs have been described, the mechanisms by which these agents modulate channel activity are largely unknown. Most calcium channel blockers exhibit state-dependence (i.e., preferential binding to certain channel conformations), but little is known about state-dependent verapamil block of T channels. We stably expressed human Ca(v)3.1 T-type channels in human embryonic kidney 293 cells and studied the state-dependence of the drug with macroscopic and gating currents. Verapamil blocked currents at micromolar concentrations at polarized potentials similar to those reported for L-type channels, although unlike for L-type currents, it did not affect current time course. The drug exhibited use-dependence and significantly slowed the apparent recovery from inactivation. Current inhibition was dependent on potential. This dependence was restricted to negative potentials, although all data were consistent with verapamil binding in the pore. Gating currents were unaffected by verapamil. We propose that verapamil achieves its inhibitory effect via occlusion of the channel pore associated with an open/inactivated conformation of the channel.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Chronic Nicotine Treatment Upregulates L-Type High Voltage-Gated Calcium Channels

Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca2+ channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [45Ca2+] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of α1 and α2/δ1 subunits for L-type HVCCs and caused an increase in the Bmax value of [3H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca2+ through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome.

Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2.

Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q) and the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.

Simultaneous Binding of Two Different Drugs in the Binding Pocket of the Human Multidrug Resistance P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) transports a wide variety of structurally diverse compounds out of the cell. The drug-binding pocket of P-gp is located in the transmembrane domains. Although occupation of the drug-binding pocket by one molecule is sufficient to activate the ATPase activity of P-gp, the drug-binding pocket may be large enough to accommodate two different substrates at the same time. In this study, we used cysteine-scanning mutagenesis to test whether P-gp could simultaneously interact with the thiol-reactive drug substrate, Tris-(2-maleimidoethyl)amine (TMEA) and a second drug substrate. TMEA is a cross-linker substrate of P-gp that allowed us to test for stimulation of cross-linking by a second substrate such as calcein-acetoxymethyl ester, colchicine, demecolcine, cyclosporin A, rhodamine B, progesterone, and verapamil. We report that verapamil induced TMEA cross-linking of mutant F343C(TM6)/V982C(TM12). By contrast, no cross-linked product was detected in mutants F343C(TM6), V982C(TM12), or F343C(TM6)/V982C(TM12) in the presence of TMEA alone. The verapamil-stimulated ATPase activity of mutant F343C(TM6)/V982C(TM12) in the presence of TMEA decreased with increased cross-linking of the mutant protein. These results show that binding of verapamil must induce changes in the drug-binding pocket (induced-fit mechanism) resulting in exposure of residues F343C(TM6)/V982C(TM12) to TMEA. The results also indicate that the common drug-binding pocket in P-gp is large enough to accommodate both verapamil and TMEA simultaneously and suggests that the substrates must occupy different regions in the common drug-binding pocket.

Drug binding analysis of human alpha 1-acid glycoprotein using capillary electrophoresis

Drug-plasma protein binding analysis is indispensable for drug development and clinical use. However, conventional methods for binding analyses were not suitable for small amounts of proteins because of large sample requirements. On the other hand, high-performance frontal analysis/capillary electrophoresis (HPFA/CE) consumes very small sample volumes, and is useful for ligand-binding study of small amounts of proteins. In this study, HPFA/CE was used in a drug-binding study of alpha 1-acid glycoprotein (AGP) subtypes in which plasma concentrations change dynamically to elucidate the effects of structural variation on drug binding. Binding study on desialyrated AGP revealed that (S)-enantiomer selectivity in propranolol-AGP binding was caused by sialic acid residues, while neither sialic acid nor galactose caused the enantioselectivity of verapamil binding to AGP. Biantennary glycans slightly suppressed disopyramide binding to AGP, whereas the glycans did not have any influence on propranolol and verapamil binding. Disopyramide and verapamil were selectively bound to the A variant rather than the F1S variant. The A variant showed larger enantioselective binding to disopyramide, but not to verapamil.

A Kinetic Evaluation of the Absorption, Efflux, and Metabolism of Verapamil in the Autoperfused Rat Jejunum

P-glycoprotein (P-gp)-mediated drug efflux from the apical membrane of enterocytes is believed to modulate intestinal cytochrome P450 3A (CYP3A) metabolism by altering substrate access to the CYP3A enzyme. This interplay between P-gp and CYP3A was investigated in a rat in situ model of intestinal permeation, where a recirculating luminal perfusion of the jejunum was coupled with mesenteric vein blood collection to simultaneously monitor the uptake, transport, and metabolism of the P-gp and CYP3A substrate, verapamil. Transport of intact verapamil into the venous blood was increased by 160, 84, and 160%, and the intestinal extraction ratio on passage across the jejunum was reduced by 15, 24, and 97% by inhibitors of P-gp [PSC833 ([3'-keto-Me-Bmt(1)]-[Val(2)]-cyclosporin)], CYP3A (midazolam), or P-gp and CYP3A (ketoconazole), respectively, when present in the luminal perfusate and compared with control experiments. Compartmental kinetic analysis of the data revealed that inhibition of P-gp did not affect the rate constant describing verapamil metabolism but, rather, increased the intestinal uptake of verapamil and stimulated a disproportionate increase in verapamil transport into the venous blood. The increase in verapamil transport, in the absence of changes to metabolism, reduced the intestinal extraction ratio. This finding may be explained by saturation of intracellular verapamil binding sites within the intestinal tissue in response to increased verapamil uptake resulting from P-gp inhibition. The current findings confirm previous in vitro and theoretical approaches which suggest that P-gp can modulate the extent of intestinal extraction of P-gp/CYP3A substrates.

Pharmacophore Model of Drugs Involved in P-Glycoprotein Multidrug Resistance: Explanation of Structural Variety (Hypothesis)

A general pharmacophore model of P-glycoprotein (P-gp) drugs is proposed that is based on a highly diverse data set and relates to the verapamil binding site of the protein. It is derived from structurally different drugs using the program GASP. The pharmacophore model consists of two hydrophobic points, three hydrogen bond (HB) acceptor points, and one HB donor point. Pharmacophore patterns of various drugs are obtained, and different binding modes are presumed for some of them. It is concluded that the binding affinity of the drugs depends on the number of the pharmacophore points simultaneously involved in the interaction with P-gp. On the basis of the obtained results, a hypothesis is proposed to explain the broad structural variety of the P-gp substrates and inhibitors: (i) the verapamil binding site of P-gp has several points that can participate in hydrophobic and HB interactions; (ii) different drugs can interact with different receptor points in different binding modes.

The use of reconstitution and inhibitor/ion interaction assays to distinguish between Ca2+/H+and Cd2+/H+antiporter activities of oat and tobacco tonoplast vesicles

Tonoplast, ion antiport activities are critical to ion homeostasis and sequestration in plants. The biochemical properties of these activities, and the enzymes that catalyse them, are little characterized. Here we applied biochemical approaches to study some characteristics and to distinguish between Ca 2+ /H + and Cd 2+ /H + antiporter activities of tonoplast vesicles from non-transformed, wild-type plants. Solubilization and reconstitution of oat-seedling (Avena sativa L.) root tonoplast vesicles resulted in about a 6-fold loss of protein, about a 6-fold enhancement of Cd 2+ /H + antiport specific activity (at 10 μM Cd 2+ ), and almost complete loss of Ca 2+ /H + antiport activity. Similar results were found for vesicles from mature tobacco (Nicotiana tabacum) roots. Cd 2+ concentration-dependent proton efflux was similar and linear with both oat vesicles and proteoliposomes. In contrast, Ca 2+ concentration-dependent proton efflux of oat vesicles was easily observed while that with proteoliposomes was minimal and nonlinear. Cd 2+ pre-treatment of oat vesicles reduced verapamil inhibition of Cd 2+ /H + activity and verapamil binding to vesicles, while Ca 2+ pre-treatment was much less protective of Ca 2+ /H + activity and verapamil binding. Results show the usefulness of reconstitution, and also inhibitor/ion interaction assays for distinguishing between transporter activities in vitro, but they do not resolve the question of whether there are separate enzymes for Cd 2+ /H + and Ca 2+ /H + . Our observation that solubilization and reconstitution have similar effects on both Cd 2+ /H + and Ca 2+ /H + activities of root tonoplast vesicles from immature oat and mature tobacco roots suggests that the transporters involved are similar in young and mature roots, and in roots of different species.

Application of three-dimensional quantitative structure-activity relationships of P-glycoprotein inhibitors and substrates.

Using in vitro data, we previously built Catalyst 3-dimensional quantitative structure activity relationship (3D-QSAR) models that qualitatively rank and predict IC(50) values for P-glycoprotein (P-gp) inhibitors. These models were derived and tested with data for inhibition of digoxin transport, calcein accumulation, vinblastine accumulation, and vinblastine binding. In the present study, 16 inhibitors of verapamil binding to P-gp were predicted using these models. These inhibition results were then used to generate a new pharmacophore that consisted of one hydrogen bond acceptor, one ring aromatic feature, and two hydrophobes. This model predicted the rank order of the four data sets described previously and correctly ranked the inhibitory potency of a further four verapamil metabolites identified in the literature. The degree of similarity in rank ordering prediction by these inhibitor pharmacophore models generated to date confirms a likely overlap in the sites to which the three P-gp substrates used in these studies (verapamil, vinblastine, and digoxin) bind. Alignment of the three substrate probes indicated that they are likely to bind the same or overlapping sites within P-gp. Important features on these substrates include multiple hydrophobic and hydrogen bond acceptor features, which are widely dispersed and in agreement among most of the five inhibitor pharmacophores we have described so far. These 3D-QSAR models will be useful for future prediction of likely substrates and inhibitors of P-gp.