Structural basis of promiscuous substrate transport by Organic Cation Transporter 1.

Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.

Atypical Substrates of the Organic Cation Transporter 1.

The human organic cation transporter 1 (OCT1) is expressed in the liver and mediates hepatocellular uptake of organic cations. However, some studies have indicated that OCT1 could transport neutral or even anionic substrates. This capability is interesting concerning protein-substrate interactions and the clinical relevance of OCT1. To better understand the transport of neutral, anionic, or zwitterionic substrates, we used HEK293 cells overexpressing wild-type OCT1 and a variant in which we changed the putative substrate binding site (aspartate474) to a neutral amino acid. The uncharged drugs trimethoprim, lamivudine, and emtricitabine were good substrates of hOCT1. However, the uncharged drugs zalcitabine and lamotrigine, and the anionic levofloxacin, and prostaglandins E2 and F2α, were transported with lower activity. Finally, we could detect only extremely weak transport rates of acyclovir, ganciclovir, and stachydrine. Deleting aspartate474 had a similar transport-lowering effect on anionic substrates as on cationic substrates, indicating that aspartate474 might be relevant for intra-protein, rather than substrate-protein, interactions. Cellular uptake of the atypical substrates by the naturally occurring frequent variants OCT1*2 (methionine420del) and OCT1*3 (arginine61cysteine) was similarly reduced, as it is known for typical organic cations. Thus, to comprehensively understand the substrate spectrum and transport mechanisms of OCT1, one should also look at organic anions.

Amino acids in transmembrane helix 1 confer major functional differences between human and mouse orthologs of the polyspecific membrane transporter OCT1.

Organic cation transporter 1 (OCT1) is a membrane transporter that affects hepatic uptake of cationic and weakly basic drugs. OCT1 transports structurally highly diverse substrates. The mechanisms conferring this polyspecificity are unknown. Here, we analyzed differences in transport kinetics between human and mouse OCT1 orthologs to identify amino acids that contribute to the polyspecificity of OCT1. Following stable transfection of HEK293 cells, we observed more than twofold differences in the transport kinetics of 22 out of 28 tested substrates. We found that the ß2-adrenergic drug fenoterol was transported with eightfold higher affinity but at ninefold lower capacity by human OCT1. In contrast, the anticholinergic drug trospium was transported with 11-fold higher affinity but at ninefold lower capacity by mouse Oct1. Using human-mouse chimeric constructs and site-directed mutagenesis, we identified nonconserved amino acids Cys36 and Phe32 as responsible for the species-specific differences in fenoterol and trospium uptake. Substitution of Cys36 (human) to Tyr36 (mouse) caused a reversal of the affinity and capacity of fenoterol but not trospium uptake. Substitution of Phe32 to Leu32 caused reversal of trospium but not fenoterol uptake kinetics. Comparison of the uptake of structurally similar ß2-adrenergics and molecular docking analyses indicated the second phenol ring, 3.3 to 4.8 Å from the protonated amino group, as essential for the affinity for fenoterol conferred by Cys36. This is the first study to report single amino acids as determinants of OCT1 polyspecificity. Our findings suggest that structure-function data of OCT1 is not directly transferrable between substrates or species.

A Multiplexed HILIC-MS/HRMS Assay for the Assessment of Transporter Inhibition Biomarkers in Phase I Clinical Trials: Isobutyryl-Carnitine as an Organic Cation Transporter (OCT1) Biomarker.

There is a growing interest in using endogenous compounds as drug transporter biomarkers to facilitate drug-drug interaction (DDI) risk assessment in early phase I clinical trials. Compared to other drug transporters, however, no valid biomarker for hepatic organic cation transporter (OCT) 1 has been described to date. The present work represents the first report of an endogenous compound, isobutyryl-l-carnitine (IBC), as a potential clinical OCT1 biomarker for DDI assessment. A hydrophilic interaction chromatography (HILIC)-mass spectrometry/high resolution mass spectrometry (MS/HRMS) assay with a simple sample preparation method was developed. The assay is capable of simultaneously quantifying multiple endogenous compounds, including IBC, thiamine, N1-methylnicotinamide (1-NMN), creatinine, carnitine, and metformin, which is a probe for OCT1 and OCT2 and MATE1 and MATE2K (multidrug and toxin extrusion proteins) in clinical studies. The HRMS assay was fit-for-purpose validated in human plasma and demonstrated good linearity, accuracy, and precision for all analytes. It was further applied to two phase I clinical trials to evaluate potential biomarkers for OCT1 and additional cation transporters (renal OCT2, MATE1, and MATE2K). The clinical data demonstrated that plasma IBC changes correlated well with in vitro data and supported its use as a liver OCT1 biomarker. The described HILIC-MS/HRMS assay can be used as a "biomarker cocktail" to simultaneously assess clinical DDI risk for the inhibition of OCT1/2 and MATEs in clinical studies with new drug candidates.

Opioids as Substrates and Inhibitors of the Genetically Highly Variable Organic Cation Transporter OCT1.

Genetic variants in the hepatic uptake transporter OCT1, observed in 9% of Europeans and white Americans, are known to affect pharmacokinetics and efficacy of tramadol, morphine, and codeine. Here, we report further opioids to be substrates and inhibitors of OCT1. Methylnaltrexone, hydromorphone, oxymorphone, and meptazinol were identified as OCT1 substrates. Methylnaltrexone is the strongest OCT1 substrate currently reported. It showed 86-fold higher accumulation in OCT1-overexpressing cells compared to control cells. We observed substantial differences in the inhibitory potency among structurally highly similar morphinan opioids (IC50 ranged from 6.4 µM for dextrorphan to 2 mM for oxycodone). The ether linkage of C4-C5 in the morphinan ring leads to a strong reduction of inhibitory potency. In conclusion, although polyspecific, OCT1 possesses a strong selectivity for its ligands. In contrast to methylnaltrexone and hydromorphone, oxycodone and hydrocodone do not interact with OCT1 and may be safer for use in individuals with genetic OCT1 deficiency.

Discovery of Competitive and Noncompetitive Ligands of the Organic Cation Transporter 1 (OCT1; SLC22A1).

Organic cation transporter 1 (OCT1) plays a critical role in the hepatocellular uptake of structurally diverse endogenous compounds and xenobiotics. Here we identified competitive and noncompetitive OCT1-interacting ligands in a library of 1780 prescription drugs by combining in silico and in vitro methods. Ligands were predicted by docking against a comparative model based on a eukaryotic homologue. In parallel, high-throughput screening (HTS) was conducted using the fluorescent probe substrate ASP+ in cells overexpressing human OCT1. Thirty competitive OCT1 ligands, defined as ligands predicted in silico as well as found by HTS, were identified. Of the 167 ligands identified by HTS, five were predicted to potentially cause clinical drug interactions. Finally, virtual screening of 29 332 metabolites predicted 146 competitive OCT1 ligands, of which an endogenous neurotoxin, 1-benzyl-1,2,3,4-tetrahydroisoquinoline, was experimentally validated. In conclusion, by combining docking and in vitro HTS, competitive and noncompetitive ligands of OCT1 can be predicted.

Molecular Properties of Drugs Interacting with SLC22 Transporters OAT1, OAT3, OCT1, and OCT2: A Machine-Learning Approach.

Statistical analysis was performed on physicochemical descriptors of ∼250 drugs known to interact with one or more SLC22 "drug" transporters (i.e., SLC22A6 or OAT1, SLC22A8 or OAT3, SLC22A1 or OCT1, and SLC22A2 or OCT2), followed by application of machine-learning methods and wet laboratory testing of novel predictions. In addition to molecular charge, organic anion transporters (OATs) were found to prefer interacting with planar structures, whereas organic cation transporters (OCTs) interact with more three-dimensional structures (i.e., greater SP3 character). Moreover, compared with OAT1 ligands, OAT3 ligands possess more acyclic tetravalent bonds and have a more zwitterionic/cationic character. In contrast, OCT1 and OCT2 ligands were not clearly distinquishable form one another by the methods employed. Multiple pharmacophore models were generated on the basis of the drugs and, consistent with the machine-learning analyses, one unique pharmacophore created from ligands of OAT3 possessed cationic properties similar to OCT ligands; this was confirmed by quantitative atomic property field analysis. Virtual screening with this pharmacophore, followed by transport assays, identified several cationic drugs that selectively interact with OAT3 but not OAT1. Although the present analysis may be somewhat limited by the need to rely largely on inhibition data for modeling, wet laboratory/in vitro transport studies, as well as analysis of drug/metabolite handling in Oat and Oct knockout animals, support the general validity of the approach-which can also be applied to other SLC and ATP binding cassette drug transporters. This may make it possible to predict the molecular properties of a drug or metabolite necessary for interaction with the transporter(s), thereby enabling better prediction of drug-drug interactions and drug-metabolite interactions. Furthermore, understanding the overlapping specificities of OATs and OCTs in the context of dynamic transporter tissue expression patterns should help predict net flux in a particular tissue of anionic, cationic, and zwitterionic molecules in normal and pathophysiological states.

Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2.

Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates--in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1-prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M(-1) s(-1) and an even slower dissociation rate (kd) in the range of 10-4 s(-1) for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1--which is normally expressed in liver and other tissues--interacts with prostaglandin analogs. While it is not entirely clear from the published literature whether OCTs function as major prostaglandin transporters, the tight binding of the naturally occurring PGE2, as well as the slow dissociation rate, could conceivably affect the transport of lower affinity substrates such as drugs and metabolites by SLC22 transporters. More research is necessary to establish the extent to which individual SLC22 family members actually function as PG transporters in vitro and in vivo and to investigate whether PGs can, independent of being directly transported, alter the ability of SLC22 transporters to handle drugs and other substrates.

Role of a Hydrophobic Pocket in Polyamine Interactions with the Polyspecific Organic Cation Transporter OCT3.

Organic cation transporter 3 (OCT3, SLC22A3) is a polyspecific, facilitative transporter expressed in astrocytes and in placental, intestinal, and blood-brain barrier epithelia, and thus elucidating the molecular mechanisms underlying OCT3 substrate recognition is critical for the rational design of drugs targeting these tissues. The pharmacology of OCT3 is distinct from that of other OCTs, and here we investigated the role of a hydrophobic cavity tucked within the translocation pathway in OCT3 transport properties. Replacement of an absolutely conserved Asp by charge reversal (D478E), neutralization (D478N), or even exchange (D478E) abolished MPP(+) uptake, demonstrating this residue to be obligatory for OCT3-mediated transport. Mutations at non-conserved residues lining the putative binding pocket of OCT3 to the corresponding residue in OCT1 (L166F, F450L, and E451Q) reduced the rate of MPP(+) transport, but recapitulated the higher sensitivity pharmacological profile of OCT1. Thus, interactions of natural polyamines (putrescine, spermidine, spermine) and polyamine-like potent OCT1 blockers (1,10-diaminodecane, decamethonium, bistriethylaminodecane, and 1,10-bisquinuclidinedecane) with wild-type OCT3 were weak, but were significantly potentiated in the mutant OCT3s. Conversely, a reciprocal mutation in OCT1 (F161L) shifted the polyamine-sensitivity phenotype toward that of OCT3. Further analysis indicated that OCT1 and OCT3 can recognize essentially the same substrates, but the strength of substrate-transporter interactions is weaker in OCT3, as informed by the distinct makeup of the hydrophobic cleft. The residues identified here are key contributors to both the observed differences between OCT3 and OCT1 and to the mechanisms of substrate recognition by OCTs in general.

Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture.

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2). 2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)>BCRP (9)>OCT1 (8)>OATP1B1 (5)>MRP3 (2). 3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2. 4. Structure-activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.

Role of the plasma membrane transporter of organic cations OCT1 and its genetic variants in modern liver pharmacology.

Changes in the uptake of many drugs by the target cells may dramatically affect the pharmacological response. Thus, downregulation of SLC22A1, which encodes the organic cation transporter type 1 (OCT1), may affect the response of healthy hepatocytes and liver cancer cells to cationic drugs, such as metformin and sorafenib, respectively. Moreover, the overall picture may be modified to a considerable extent by the preexistence or the appearance during the pathogenic process of genetic variants. Some rare OCT1 variants enhance transport activity, whereas other more frequent variants impair protein maturation, plasma membrane targeting or the function of this carrier, hence reducing intracellular active drug concentrations. Here, we review current knowledge of the role of OCT1 in modern liver pharmacology, which includes the use of cationic drugs to treat several diseases, some of them of great clinical relevance such as diabetes and primary liver cancer (cholangiocarcinoma and hepatocellular carcinoma). We conclude that modern pharmacology must consider the individual evaluation of OCT1 expression/function in the healthy liver and in the target tissue, particularly if this is a tumor, in order to predict the lack of response to cationic drugs and to be able to design individualized pharmacological treatments with the highest chances of success.

Functional analysis of novel variants in the organic cation/ergothioneine transporter 1 identified in Singapore populations.

The human organic cation/ergothioneine transporter 1 (hOCTN1, gene symbol SLC22A4) is responsible for the cellular uptake of substances, such as L-ergothioneine, which is an important antioxidant in mammalian cells. The common-function-altered variant L503F-hOCTN1 has been associated with susceptibility to Crohn's disease in certain populations. Previously, we identified eight novel nonsynonymous single-nucleotide polymorphisms (SNPs) in the SLC22A4 gene in the Chinese and Indian populations of Singapore. The present study evaluated the impact of these novel SNPs on hOCTN1 transport function in HEK-293 cells. Transport uptake assays with L-ergothioneine were used to assess the function of the variant transporters. Cell surface biotinylation and Western blot analysis were used to characterize cellular transporter expression. Comparative modeling was used to locate amino acid substitutions in the topology of hOCTN1 in order to account for altered transport function. Transporter activity was markedly impaired in four of the naturally occurring hOCTN1 variants (R63H, R83P, G482D, and I500N). Multiple glycosylated isoforms of hOCTN1 proteins were identified in the plasma membrane and in the whole cell. Either the total cellular or membrane expression of the functionally deficient transporter variants was lower than that of the wild-type hOCTN1. The underlying mechanism involves both impaired transporter-substrate binding affinity and turnover rate. Considered together, several naturally occurring SNPs in the SLC22A4 gene encode variant hOCTN1 transporters that may impact the cellular uptake of L-ergothioneine and other substrates, with the potential to influence the antioxidant capacity of human cells.

Expression of SLC22A1 variants may affect the response of hepatocellular carcinoma and cholangiocarcinoma to sorafenib.

UNLABELLED: Reduced drug uptake is an important mechanism of chemoresistance. Down-regulation of SLC22A1 encoding the organic cation transporter-1 (OCT1) may affect the response of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CGC) to sorafenib, a cationic drug. Here we investigated whether SLC22A1 variants may contribute to sorafenib chemoresistance. Complete sequencing and selective variant identification were carried out to detect single nucleotide polymorphisms (SNPs) in SLC22A1 complementary DNA (cDNA). In HCC and CGC biopsies, in addition to previously described variants, two novel alternative spliced variants and three SNPs were identified. To study their functional consequences, these variants were mimicked by directed mutagenesis and expressed in HCC (Alexander and SK-Hep-1) and CGC (TFK1) cells. The two novel described variants, R61S fs*10 and C88A fs*16, encoded truncated proteins unable to reach the plasma membrane. Both variants abolished OCT1-mediated uptake of tetraethylammonium, a typical OCT1 substrate, and were not able to induce sorafenib sensitivity. In cells expressing functional OCT1 variants, OCT1 inhibition with quinine prevented sorafenib-induced toxicity. Expression of OCT1 variants in Xenopus laevis oocytes and determination of quinine-sensitive sorafenib uptake by high-performance liquid chromatography-dual mass spectrometry confirmed that OCT1 is able to transport sorafenib and that R61S fs*10 and C88A fs*16 abolish this ability. Screening of these SNPs in 23 HCC and 15 CGC biopsies revealed that R61S fs*10 was present in both HCC (17%) and CGC (13%), whereas C88A fs*16 was only found in HCC (17%). Considering all SLC22A1 variants, at least one inactivating SNP was found in 48% HCC and 40% CGC. CONCLUSION: Development of HCC and CGC is accompanied by the appearance of aberrant OCT1 variants that, together with decreased OCT1 expression, may dramatically affect the ability of sorafenib to reach active intracellular concentrations in these tumors.

The hOCT1 SNPs M420del and M408V alter imatinib uptake and M420del modifies clinical outcome in imatinib-treated chronic myeloid leukemia.

Although the prognosis of chronic myeloid leukemia (CML) patients treated with imatinib is good, many fail to develop an optimal response or lose one. This heterogeneity could be attributed to the presence of human organic cation transporter-1 (hOCT1) single nucleotide polymorphisms (SNPs). In the present study, we analyzed the effect of 23 hOCT1 SNPs on imatinib treatment outcome in newly diagnosed CML patients using MassARRAY sequencing and pyrosequencing. The only SNP associated with outcome was M420del (rs35191146), with patients with the M420del demonstrating an increased probability of imatinib treatment failure. In CML cell lines transfected with M420del and/or M408V, M420del significantly decreased imatinib uptake, but this effect was countered if the M408V (rs628031) SNP was also present. A similar effect was seen for the uptake of the hOCT1 substrates TEA(+) and ASP(+). Finally, apparent hOCT1 mRNA levels were studied using both our earlier primers covering the M420del and another set that did not. Different mRNA expression was observed, explaining the disparity in published data on the prognostic importance of hOCT1 mRNA and highlighting the importance of avoiding common SNP sites in primer design. These data demonstrate that the common M420del SNP can modulate the outcome of imatinib treatment.

Impact of protein/protein interactions on global intermolecular translocation rates of the transcription factors Sox2 and Oct1 between DNA cognate sites analyzed by z-exchange NMR spectroscopy.

Oct1 and Sox2 synergistically regulate developmental genes by binding to adjacent sites within promoters. We have investigated the kinetics of global intermolecular translocation of Sox2 and Oct1 between cognate sites located on different DNA molecules by z-exchange NMR spectroscopy. In the Hoxb1 promoter, the Sox2 and Oct1 sites are immediately adjacent to one another, and the intermolecular translocation rates are too slow to be measured by z-exchange spectroscopy. By introducing a 3-bp insertion between the Sox2 and Oct1 sites to mimic the spacing in the FGF4 enhancer, the interprotein contact surface is reduced, and the translocation rates are increased. Interaction between Sox2 and the POU-specific domain (POU(S)) of Oct1 does not affect the translocation mechanism but modulates the rates. Translocation involves only jumping (dissociation and reassociation) for Sox2, but both jumping and direct intersegment transfer (no dissociation into free solution) for Oct1. The dissociation (k(off) ∼1.5 s(-1)) and association (k(on) ∼5.1 × 10(9) m(-1)s(-1)) rate constants for Sox2 are reduced 4-fold and increased 5-fold, respectively, in the presence of Oct1. k(off) (∼3.5 s(-1)) for Oct1 is unaffected by Sox2, whereas k(on) (∼1.3 × 10(9) m(-1)s(-1)) is increased ∼13-fold. The direct intermolecular translocation rate (k(inter) ∼1.8 × 10(4) m(-1)s(-1)) for the POU(S) domain of Oct1 is reduced 2-fold by Sox2, whereas that for the POU homeodomain (POU(HD)) of Oct1 (k(inter) ∼ 1.7 × 10(4) m(-1)s(-1)) remains unaltered, consistent with the absence of contacts between Sox2 and POU(HD). The data suggest a model for the sequence of binding events involved in synergistic gene regulation by Sox2 and Oct1.

Interplay between minor and major groove-binding transcription factors Sox2 and Oct1 in translocation on DNA studied by paramagnetic and diamagnetic NMR.

The pathways whereby Sox2 scans DNA to locate its specific binding site are investigated by NMR in specific and nonspecific Sox2·DNA complexes and in a specific ternary complex with Oct1 on the Hoxb1 regulatory element. Direct transfer of Sox2 between nonspecific sites on different DNA molecules occurs without dissociation into free solution at a rate of ∼10(6) M(-1) s(-1), whereas one-dimensional sliding proceeds with a diffusion constant of ≥0.1 µm(2)·s(-1). Translocation of Sox2 from one specific DNA site to another occurs via jumping, involving complete dissociation into free solution (k(d) ∼5-6 s(-1)) followed by reassociation (k(a) ∼5 × 10(8) M(-1) s(-1)). In the presence of Oct1 bound to an adjacent specific site, k(d) is reduced by more than 10-fold. Paramagnetic relaxation measurements, however, demonstrate that sparsely populated (<1%), transient states involving nonspecifically bound Sox2 in rapid exchange with specifically bound Sox2 are sampled in both binary Sox2·DNA- and ternary Oct1·Sox2·Hoxb1-DNA-specific complexes. Moreover, Sox2 modulates the mechanism of translocation of Oct1. Both Sox2 and the Oct1 POU(HD) domain are transiently released from the specific ternary complex by sliding to an adjacent nonspecific site, followed by direct transfer to another DNA molecule, whereas the Oct1 POU(S) domain is fixed to its specific site through direct interactions with Sox2. Intermolecular translocation of POU(HD) results in the formation of a bridged intermediate spanning two DNA molecules, enhancing the probability of complete intermolecular translocation of Oct1. By way of contrast, in the specific Oct1·DNA binary complex, POU(S) undergoes direct intermolecular translocation, whereas POU(HD) scans the DNA by sliding.

Intra- and intermolecular translocation of the bi-domain transcription factor Oct1 characterized by liquid crystal and paramagnetic NMR.

The intra- and intermolecular translocation processes whereby the bi-domain transcription factor Oct1 searches for its specific DNA target site have been investigated by residual dipolar coupling (RDC) and paramagnetic relaxation enhancement (PRE) measurements. The RDC data show that the orientation of the POU(S) and POU(HD) domains of Oct1 relative to the long axis of the DNA is the same for specific and nonspecific complexes with DNA. In the context of the specific Oct1-DNA complex, sparsely-populated, spectroscopically "invisible" states reveal their footprints on the PRE profiles observed for the specific complex. Analysis of the PRE data indicates that the POU(HD) domain searches the DNA primarily by rotation-coupled sliding (intramolecular translocation), while the POU(S) domain functions as an antenna to promote intersegment transfer via intermolecular translocation. The latter involves the formation of a bridged intermediate in which the POU(HD) domain is located on the first DNA molecule and the POU(S) domain on the second. The formation of the bridge intermediate promotes the completion of intermolecular translocation of Oct1 via a first order process involving dissociation and association of the POU(HD) domain from one DNA molecule to another. Thus cross-talk between the POU(S) and POU(HD) domains, each fulfilling different and complementary components of the search process ensures efficient sampling of DNA, thereby facilitating the location of specific Oct1 target sites.

Tethered-hopping model for protein-DNA binding and unbinding based on Sox2-Oct1-Hoxb1 ternary complex simulations.

The sliding and hopping models encapsulate the essential protein-DNA binding process for binary complex formation and dissociation. However, the effects of a cofactor protein on the protein-DNA binding process that leads to the formation of a ternary complex remain largely unknown. Here we investigate the effect of the cofactor Sox2 on the binding and unbinding of Oct1 with the Hoxb1 control element. We simulate the association of Oct1 with Sox2-Hoxb1 using molecular dynamics simulations, and the dissociation of Oct1 from Sox2-Hoxb1 using steered molecular dynamics simulations, in analogy to a hopping event of Oct1. We compare the kinetic and thermodynamic properties of three model complexes (the wild-type and two mutants) in which the Oct1-DNA base-specific interactions or the Sox2-Oct1 protein-protein interactions are largely abolished. We find that Oct1-DNA base-specific interactions contribute significantly to the total interaction energy of the ternary complex, and that nonspecific Oct1-DNA interactions are sufficient for driving the formation of the protein-DNA interface. The Sox2-Oct1 protein-protein binding interface is largely hydrophobic, with remarkable shape complementarity. This interface promotes the formation of the ternary complex and slows the dissociation of Oct1 from its DNA-binding site. We propose a simple two-step reaction model of protein-DNA binding, called the tethered-hopping model, that explains the importance of the cofactor Sox2 and may apply to similar ternary protein-DNA complexes.

Organic cation transporters and their roles in antiretroviral drug disposition.

BACKGROUND: Understanding metabolising processes and drug-transporter interactions is particularly crucial to the management of patients with HIV infection, given the several antiretroviral drugs that must be taken lifelong and the use of other medications for HIV-related and non-HIV-related conditions. Several interactions of antiretroviral drugs with metabolising enzymes, especially cytochrome P450 and ATP-dependent transporter P-glycoprotein, have been described but the role of the organic cation transporters (OCTs) is less clearly defined. OBJECTIVE: To review the relevance of the OCTs for antiretroviral drug disposition. METHODS: Interactions of OCTs with antiretroviral drugs and evidence for clinical relevance are discussed. RESULTS/CONCLUSION: Several antiretroviral drugs show relevant interactions with the OCTs in cell-based experiments and the OCTs are highly upregulated in HIV-infected patients. For evaluating the clinical significance, interaction studies in HIV patients and reliable in vitro models for delineation of in vivo effects are needed.