A High-Throughput Screen of a Library of Therapeutics Identifies Cytotoxic Substrates of P-glycoprotein.

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action. We used the CellTiter-Glo viability assay to test library compounds against parental KB-3-1 human cervical adenocarcinoma cells and the colchicine-selected subline KB-8-5-11 that overexpresses P-gp. KB-8-5-11 cells were also tested in the presence of a P-gp inhibitor (tariquidar) to assess reversibility of transporter-mediated resistance. Of the tested compounds, a total of 90 P-gp substrates were identified, including 55 newly identified compounds. Substrates were confirmed using an orthogonal killing assay against human embryonic kidney-293 cells overexpressing P-gp. We confirmed that AT7159 (cyclin-dependent kinase inhibitor), AT9283, (Janus kinase 2/3 inhibitor), ispinesib (kinesin spindle protein inhibitor), gedatolisib (PKI-587, phosphoinositide 3-kinase/mammalian target of rampamycin inhibitor), GSK-690693 (AKT inhibitor), and KW-2478 (heat-shock protein 90 inhibitor) were substrates. In addition, we assessed direct ATPase stimulation. ABCG2 was also found to confer high levels of resistance to AT9283, GSK-690693, and gedatolisib, whereas ispinesib, AT7519, and KW-2478 were weaker substrates. Combinations of P-gp substrates and inhibitors were assessed to demonstrate on-target synergistic cell killing. These data identified compounds whose oral bioavailability or brain penetration may be affected by P-gp. SIGNIFICANCE STATEMENT: The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to be expressed at barrier sites, where it acts to limit oral bioavailability and brain penetration of substrates. In order to identify novel compounds that are transported by P-gp, we developed a high-throughput screen using the KB-3-1 cancer cell line and its colchicine-selected subline KB-8-5-11. We screened the Mechanism Interrogation Plate (MIPE) library, the National Center for Advancing Translational Science (NCATS) pharmaceutical collection (NPC), the NCATS Pharmacologically Active Chemical Toolbox (NPACT), and a kinase inhibitor library comprising 977 compounds, for a total of 10,804 compounds. Of the 10,804 compounds screened, a total of 90 substrates were identified of which 55 were novel. P-gp expression may adversely affect the oral bioavailability or brain penetration of these compounds.

Searching for New Leads To Treat Epilepsy: Target-Based Virtual Screening for the Discovery of Anticonvulsant Agents.

The purpose of this investigation is to contribute to the development of new anticonvulsant drugs to treat patients with refractory epilepsy. We applied a virtual screening protocol that involved the search into molecular databases of new compounds and known drugs to find small molecules that interact with the open conformation of the Nav1.2 pore. As the 3D structure of human Nav1.2 is not available, we first assembled 3D models of the target, in closed and open conformations. After the virtual screening, the resulting candidates were submitted to a second virtual filter, to find compounds with better chances of being effective for the treatment of P-glycoprotein (P-gp) mediated resistant epilepsy. Again, we built a model of the 3D structure of human P-gp, and we validated the docking methodology selected to propose the best candidates, which were experimentally tested on Nav1.2 channels by patch clamp techniques and in vivo by the maximal electroshock seizure (MES) test. Patch clamp studies allowed us to corroborate that our candidates, drugs used for the treatment of other pathologies like Ciprofloxacin, Losartan, and Valsartan, exhibit inhibitory effects on Nav1.2 channel activity. Additionally, a compound synthesized in our lab, N, N'-diphenethylsulfamide, interacts with the target and also triggers significant Na1.2 channel inhibitory action. Finally, in vivo studies confirmed the anticonvulsant action of Valsartan, Ciprofloxacin, and N, N'-diphenethylsulfamide.

Docking applied to the prediction of the affinity of compounds to P-glycoprotein.

P-glycoprotein (P-gp) is involved in the transport of xenobiotic compounds and responsible for the decrease of the drug accumulation in multi-drug-resistant cells. In this investigation we compare several docking algorithms in order to find the conditions that are able to discriminate between P-gp binders and nonbinders. We built a comprehensive dataset of binders and nonbinders based on a careful analysis of the experimental data available in the literature, trying to overcome the discrepancy noticeable in the experimental results. We found that Autodock Vina flexible docking is the best choice for the tested options. The results will be useful to filter virtual screening results in the rational design of new drugs that are not expected to be expelled by P-gp.

Several new diverse anticonvulsant agents discovered in a virtual screening campaign aimed at novel antiepileptic drugs to treat refractory epilepsy.

A virtual screening campaign was conducted in order to discover new anticonvulsant drug candidates for the treatment of refractory epilepsy. To this purpose, a topological discriminant function to identify antiMES drugs and a sequential filtering methodology to discriminate P-glycoprotein substrates and nonsubstrates were jointly applied to ZINC 5 and DrugBank databases. The virtual filters combine an ensemble of 2D classifiers and docking simulations. In the light of the results, 10 structurally diverse compounds were acquired and tested in animal models of seizure and the rotorod test. All 10 candidates showed some level of protection against MES test.

Interaction of LDS-751 with the drug-binding site of P-glycoprotein: a Trp fluorescence steady-state and lifetime study.

P-glycoprotein (ABCB1) is an ATP-driven efflux pump which binds drugs within a large flexible binding pocket. Intrinsic Trp fluorescence was used to probe the interactions of LDS-751 (2-[4-(4-[dimethylamino]phenyl)-1,3-butadienyl]-3-ethylbenzo-thiazolium perchlorate) with purified P-glycoprotein, using steady-state/lifetime measurements and collisional quenching. The fast decay component of P-glycoprotein intrinsic fluorescence (tau(1)=0.97 ns) was unaffected by LDS-751 binding, while the slow decay component (tau(2)=4.02 ns) was quenched by dynamic and static mechanisms. Both the wavelength-dependence of the decay kinetics, and the time-resolved emission spectra, suggested the existence of excited-state relaxation processes within the protein matrix on the nanosecond time-scale, which were altered by LDS-751 binding. The fast decay component, which is more solvent-exposed, can be attributed to cytosolic/extracellular Trp residues, while the slow decay component likely arises from more buried transmembrane Trp residues. Interaction of a drug with the binding pocket of P-glycoprotein thus affects its molecular structure and fast dynamics.

Plasmodium vivax: allele variants of the mdr1 gene do not associate with chloroquine resistance among isolates from Brazil, Papua, and monkey-adapted strains.

We describe here the sequence of the Plasmodium vivax mdr1 gene from 10 different isolates differing in chloroquine sensitivity. The deduced amino acid sequence of PvMDR1 shares more than 70% similarity with other malarial MDR proteins and it displays consensus motifs of an ABC family transporter including two transmembrane domains and two ATP binding cassettes. Similarity and dendrogram analyses revealed that sequences could be grouped according to their geographical origin. Within each geographical group however, no correlation was found between chloroquine resistance and specific mutations.

Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site.

One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the existence of at least two binding sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R sites for their preference for Hoechst 33342 and rhodamine 123, respectively. In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R site. A 50-fold enhancement of LDS-751 fluorescence indicated that the protein binding site was located in a hydrophobic environment, with a polarity lower than that of chloroform. LDS-751 bound with sub-micromolar affinity (K(d) = 0.75 microM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-binding regions of the transporter and may interact directly with the dye. Using a FRET approach, we mapped the possible locations of the LDS-751 binding site relative to the NB domain active sites. The R site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet. The location of both H and R drug binding sites is in agreement with the idea that Pgp may operate as a drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane.

Cinética del enlazamiento de H33342 a la p-glicoproteína: detección y análisis de curvas de progreso / Kinetic of the binding of H33342 to the p-glycoprotein: detection and analysis of progress curves

El transportador multidrogas P-glicoproteína (Pgp) lleva a cabo el eflujo celular, ATP-dependiente, de muchas drogas hidrofóbicas, productos naturales y péptidos. Se propone que la Pgp contiene dos sitios de transporte, conocidos como los sitios -H y -R por sus preferencias por Hoechst 33342 (H33342) y rodamina 123, respectivamente. Cuando H33342 interactúa con la Pgp purificada, su rendimiento cuántico incrementa debido al ambiente hidrofóbico del bolsillo de enlazamiento –H. En este trabajo, estudiamos el enlazamiento de H33342 a la Pgp empleando experimentos cinéticos en la modalidad de stoppedflow. El curso temporal de la reacción fue seguido por el incremento de la fluorescencia del colorante y analizado con la herramienta computacional DYNAFIT, usando un modelo donde una reacción bimolecular rápida, es seguida por tres isomerizaciones secuenciales. Adicionalmente, bajo condiciones de seudo-primer-orden (exceso del ligando), la reacción presentó cuatro relajaciones caracterizadas por cuatro constantes de tiempo (á`s) y cuatro amplitudes (A¡`s) Estos parámetros fueron analizados utilizando la técnica de la matriz de los operadores de proyección. Esta aproximación aportó, por primera vez, información acerca de las constantes de velocidad y propiedades fluorescentes de los diversos intermediarios formados durante el enlazamiento de H33342 a la Pgp.

The P-glycoprotein multidrug transporter (Pgp) carries out ATP-driven cellular efflux of many different hydrophobic drugs, natural products, and peptides. Pgp is proposed to contain two drug transport sites, known as the H site and the R site for their preference for Hoechst 33342 (H33342) and rhodamine 123, respectively. When H33342 interacts with purified Pgp, its quantum yield is increased due to transfer to a hydrophobic environment within the H binding pocket, as shown by the steady-state fluorescence emission. In this work, we studied the binding of H33342 to Pgp using stopped-flow kinetic experiments. The time course of the reaction was followed by enhancement of dye fluorescence and analyzed by the computational tool DYNAFIT, using the model of a fast bimolecular reaction, followed by a three-step sequential isomerization. Additionally, under pseudo-first-order conditions (excess ligand), the reaction presented five normal modes, characterized by four relaxation times (á`s) and four amplitudes (A¡`s) These parameters were analyzed using the matrix projection operator technique, considering a four-step sequential reaction. This approach provides, for the first time, information about the rate constants and fluorescent properties of the diverse intermediates formed during the binding of H33342 to Pgp.

Multidrug resistance proteins in tuberous sclerosis and refractory epilepsy.

Tuberous sclerosis is an autosomal dominant syndrome characterized by seizures that are refractory to medication in severely affected individuals. The mechanism involved in drug resistance in tuberous sclerosis is unknown. The proteins MDR-1 (multidrug resistance) and MRP-1 (multidrug resistance-associated protein-1) are linked to chemotherapy resistance in tumor cells. However, the relationship between refractoriness to antiepileptic drugs and MDR-1 or MRP-1 brain expression has been poorly studied. We have previously described a case of tuberous sclerosis with refractory epilepsy that expressed multidrug resistance gene (MDR-1) in tuber cells from epileptogenic brain lesion. In this retrospective study, we describe the expression of MDR-1 and MRP-1 in the epileptogenic cortical tubers of three pediatric patients with tuberous sclerosis and refractory epilepsy surgically treated. Monoclonal antibodies for MDR-1 and MRP-1 proteins were used for immunohistochemistry. In epileptogenic cortical tuber brain specimens, MDR-1 and MRP-1 proteins were strongly immunoreactive in abnormal balloon cells, dysplastic neurons, astrocytes, microglial cells, and some blood-brain vessels. A more extensive MDR-1 immunoreactivity was observed. These data suggest that refractory epilepsy phenotype in tuberous sclerosis can be associated with the expression of both multidrug resistance MDR-1 and MRP-1 transporters in epileptogenic cortical tubers.

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships.

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.