Overlapping Substrate Specificities Research Articles

Genetic Heterogeneity of SLC22 Family of Transporters in Drug Disposition.

An important aspect of modern medicine is its orientation to achieve more personalized pharmacological treatments. In this context, transporters involved in drug disposition have gained well-justified attention. Owing to its broad spectrum of substrate specificity, including endogenous compounds and xenobiotics, and its strategical expression in organs accounting for drug disposition, such as intestine, liver and kidney, the SLC22 family of transporters plays an important role in physiology, pharmacology and toxicology. Among these carriers are plasma membrane transporters for organic cations (OCTs) and anions (OATs) with a marked overlap in substrate specificity. These two major clades of SLC22 proteins share a similar membrane topology but differ in their degree of genetic variability. Members of the OCT subfamily are highly polymorphic, whereas OATs have a lower number of genetic variants. Regarding drug disposition, changes in the activity of these variants affect intestinal absorption and target tissue uptake, but more frequently they modify plasma levels due to enhanced or reduced clearance by the liver and secretion by the kidney. The consequences of these changes in transport-associated function markedly affect the effectiveness and toxicity of the treatment in patients carrying the mutation. In solid tumors, changes in the expression of these transporters and the existence of genetic variants substantially determine the response to anticancer drugs. Moreover, chemoresistance usually evolves in response to pharmacological and radiological treatment. Future personalized medicine will require monitoring these changes in a dynamic way to adapt the treatment to the weaknesses shown by each tumor at each stage in each patient.

Dimerization of ADARs Expands The Range of Substrates That Can Undergo A‐to‐I RNA Editing

RNA editing alters genomically encoded information to generate the transcriptomic diversity required for normal development and proper neuronal function. The adenosine deaminase that act on RNA (ADAR) family of enzymes catalyze adenosine (A)‐to‐inosine (I) RNA editing at millions of sites in the human transcriptome. However, the molecular mechanisms that regulate editing specificity and efficiency are not understood. Using recombinant proteins, we reveal, that unlike all other A‐to‐I editing enzymes, Caenorhabditis elegans ADR‐2 has a low affinity for double‐stranded RNAs (dsRNAs). Using both in vivo and in vitro approaches, our data indicates that ADR‐2 can physically interact with the dsRNA binding protein, ADR‐1, which is also a deaminase‐deficient member of the ADAR family. Our data from RNA‐immunoprecipitation studies of ADR‐2 indicate that ADR‐1 is required for ADR‐2 to stably interact with some target mRNAs in vivo. Furthermore, biochemical editing assays from lysates expressing ADR‐1 mutant proteins indicate that both RNA binding by ADR‐1 and the protein‐protein interaction between ADR‐1 and ADR‐2 promotes RNA editing efficiency. In sum, we have identified a molecular mechanism for regulating the ability of ADAR to both bind target mRNAs and edit. As human ADARs are known to have some overlapping substrate specificities, these results raise the possibility that dimerization between ADAR family members, and perhaps protein‐protein interactions of ADARs with other dsRNA binding proteins, provides the A‐to‐I editing enzymes with a molecular mechanism to specifically recognize editing targets over the thousands of RNAs present in a cell.Support or Funding InformationAmerican Cancer Society Research Scholar Grant (15‐051‐RMC) to H.A.H. Indiana University School of Medicine Start‐up Funds to H.A.H.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fluorogenic structure activity library pinpoints molecular variations in the substrate specificity of structurally homologous esterases

Ubiquitous cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. These diverse substrates and functions however complicate a priori prediction of their substrate preferences and biological functions. Analogous to substrate activity screening for serine protease activity, we designed a 36‐member structure activity relationship (SAR) library of fluorogenic esterase substrates with low background hydrolysis, high sensitivity, and modular, straightforward synthesis. In three parallel substrate series containing alkyl, ether, and thioether substituents, the SAR library systemically interrogates esterase preference for chain length, branching patterns, polarity, and hydrogen bonding to differentiate common classes of esterase substrates. Applying this library against two structurally homologous bacterial esterases, previously broad overlapping substrate specificity was refined to preferences for γ‐position thioethers and ethers for ybfF from Vibrio cholerae and branched substrates with and without thioethers for Rv0045c from Mycobacterium tuberculosis. Structural control over this substrate differentiation was then assigned to individual substrate selectivity residues of Tyr116 in ybfF and His187 in Rv0045c whose reciprocal substitution inverted each esterase's substrate preference. This SAR esterase library could have multi‐faceted future applications including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health (NIH 1 R15 GM110641‐01A1).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

Kinetic Control of Quorum Sensing in Pseudomonas aeruginosa by Multidrug Efflux Pumps.

Pseudomonas aeruginosa is an important human pathogen, the physiology and virulence of which are under the control of quorum sensing signals. These signals often have dual roles, functioning as toxins to some cells and as oxidative-stress protectors for their producer cells. Hence, their internal and external concentrations should be tightly controlled. In this study, we analyzed the interplay between the multidrug efflux transporters MexEF-OprN and MexG/HI-OpmD in quorum sensing of P.aeruginosa. We found that the two transporters have overlapping substrate specificities but different efficiencies. When overproduced, both MexEF-OprN and MexG/HI-OpmD provide clinical levels of resistance to diverse fluoroquinolones and protect P.aeruginosa against toxic phenazines. However, this similarity is enabled by synergistic interactions with the outer membrane. In hyperporinated cells, MexG/HI-OpmD is saturated by much lower concentrations of fluoroquinolones but is more efficient than MexEF-OprN in efflux of phenazines. Unlike MexEF-OprN, mutational inactivation of MexG/HI-OpmD reduces the levels of pyocyanin and makes P.aeruginosa cells hypersusceptible to phenazines. Our results further show that MexG binds pyocyanin, physically associates with MexHI, and represses the activity of the transporter, revealing a negative regulatory role of this protein. We conclude that differences in kinetic properties of transporters are critical to maintain proper intra- and extracellular concentrations of phenazines and other signaling molecules and that MexG/HI-OpmD controls the steady state in the synthesis and secretion of phenazines.

Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity.

Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4-P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1' position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4-P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4-8 weeks (HyCoSuL synthesis: 3-5 weeks; HyCoSuL screening per enzyme: 4-8 d; and activity-based probe synthesis: 1-2 weeks).

Tyrphostin RG14620 selectively reverses ABCG2-mediated multidrug resistance in cancer cell lines

The multidrug resistance (MDR) phenotype associated with the overexpression of ATP-binding cassette (ABC) drug transporters ABCB1, ABCC1 and ABCG2 is a major obstacle in cancer chemotherapy. Numerous epidermal growth factor receptor (EGFR) inhibitors have previously been shown capable of reversing MDR in ABCG2-overexpressing cancer cells. However, most of them are not transporter-specific due to the substantial overlapping substrate specificity among the transporters. In this study, we investigated the interaction between ABCG2 and tyrphostin RG14620, an EGFR inhibitor of the tyrphostin family, in multidrug-resistant cancer cell lines. We found that at nontoxic concentrations, tyrphostin RG14620 enhances drug-induced apoptosis and restores chemosensitivity to ABCG2-overexpressing multidrug-resistant cancer cells. More importantly, tyrphostin RG14620 is selective to ABCG2 relative to ABCB1 and ABCC1. Our findings were further supported by biochemical assays demonstrating that tyrphostin RG14620 stimulates ATP hydrolysis and inhibits photoaffinity labeling of ABCG2 with IAAP, and by a docking analysis of tyrphostin RG14620 in the drug-binding pocket of this transporter. Taken together, our findings indicate that tyrphostin RG14620 is a potent and selective modulator of ABCG2 that may be useful to overcome chemoresistance in patients with drug-resistant tumors.

In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases

BackgroundP450 fatty acid decarboxylases represented by the unusual CYP152 peroxygenase family member OleTJE have been receiving great attention recently since these P450 enzymes are able to catalyze the simple and direct production of 1-alkenes for potential applications in biofuels and biomaterials. To gain more mechanistic insights, broader substrate spectra, and improved decarboxylative activities, it is demanded to discover and investigate more P450 fatty acid decarboxylases.ResultsHere, we describe for the first time the expression, purification, and in vitro biochemical characterization of two new CYP152 peroxygenases, CYP-Aa162 and CYP-Sm46Δ29, that are capable of decarboxylating straight-chain saturated fatty acids. Both enzymes were found to catalyze the decarboxylation and hydroxylation of a broad range of free fatty acids (C10–C20) with overlapping substrate specificity, yet distinct chemoselectivity. CYP-Sm46Δ29 works primarily as a fatty (lauric) acid decarboxylase (66.1 ± 3.9% 1-undecene production) while CYP-Aa162 more as a fatty (lauric) acid hydroxylase (72.2 ± 0.9% hydroxy lauric acid production). Notably, the optical spectroscopic analysis of functional CYP-Sm46Δ29 revealed no characteristic P450 band, suggesting a unique heme coordination environment. Active-site mutagenesis analysis showed that substitution with the proposed key decarboxylation-modulating residues, His85 and Ile170, enhanced the decarboxylation activity of CYP-Aa162 and P450BSβ, emphasizing the importance of these residues in directing the decarboxylation pathway. Furthermore, the steady-state kinetic analysis of CYP-Aa162 and CYP-Sm46Δ29 revealed both cooperative and substrate inhibition behaviors which are substrate carbon chain length dependent.ConclusionsOur data identify CYP-Sm46Δ29 as an efficient OleTJE-like fatty acid decarboxylase. Oxidative decarboxylation chemoselectivity of the CYP152 decarboxylases is largely dependent upon the carbon chain length of fatty acid substrates and their precise positioning in the enzyme active site. Finally, the kinetic mode analysis of the enzymes could provide important guidance for future process design.

Generation of AMBER force field parameters for zinc centres of M1 and M17 family aminopeptidases

The M1 and M17 aminopeptidases are metallo-exopeptidases that rely on the presence of divalent cations, usually zinc, in their active site for proteolytic activity. They are from separate protease superfamilies, however, members often have overlapping substrate specificity. Inhibitors of one or both enzymes can be used to modulate hypertension, reduce proliferation of certain types of cancers and control malaria parasites. Current inhibitors act to chelate the zinc ions in the active site, locking the enzymes in an inactive transition state. We were interested in using a computational approach to understand the structure and dynamics of the M1 and M17 aminopeptidases, however, the presence of the essential metal ions in the proteases presents a challenge to classical molecular dynamics (MD) simulation. The zinc amber force field does not contain applicable descriptions of the zinc coordination environment present in either of these two protease families. To provide tools for the study of these two enzymes, we have used the metal centre parameter builder to generate new hybrid bonded/nonbonded force field (FF) parameters to correctly describe the active site architecture for each enzyme. The new parameters were evaluated by fitting the normal mode frequencies of molecular mechanics to the quantum mechanics frequencies and validated by performing short MD simulations. The new FF parameters now enable more accurate and reliable MD simulations for any member of the M1 or M17 aminopeptidase superfamilies.

Evaluation of expression and glycosylation status of UGT1A10 in Supersomes and intestinal epithelial cells with a novel specific UGT1A10 monoclonal antibody.

UDP-Glucuronosyltransferases (UGTs) are major phase II drug-metabolizing enzymes. Each member of the UGT family exhibits a unique but occasionally overlapping substrate specificity and tissue-specific expression pattern. Earlier studies have reported that human UGT1A10 is expressed in the gastrointestinal tract at the mRNA level, but the evaluation at the protein level, especially tissue or cellular localization, has lagged behind because of the lack of a specific antibody. In this study, we prepared a monoclonal antibody to UGT1A10 to elucidate the tissue/cellular distribution and interindividual variability of UGT1A10 protein expression. Western blot analysis revealed that the prepared antibody does not cross-react with any other human UGTs. Using this specific antibody, we observed that UGT1A10 protein is expressed in the small intestine but not in the liver or kidney. Immunohistochemical analysis revealed the expression of UGT1A10 protein in epithelial cells of the crypts and villi of the duodenum. In the small intestine microsomes from six individuals, the UGT1A10 protein levels exhibited 16-fold variability. Dopamine 3- and 4-glucuronidation, which is mainly catalyzed by UGT1A10 and by other UGT isoforms marginally, exhibited 50- to 65-fold variability, and they were not correlated with the UGT1A10 protein levels. Interestingly, the enzymatic activities of recombinant UGT1A10 in insect cells that were normalized to the UGT1A10 protein level were markedly lower than those in pooled human small intestine microsomes. Thus, the UGT1A10 antibody we generated made it possible to investigate the tissue/cellular distribution and interindividual variability of UGT1A10 protein expression for understanding the pharmacological and toxicological role of UGT1A10.

No cancer predisposition or increased spontaneous mutation frequencies in NEIL DNA glycosylases-deficient mice

Base excision repair (BER) is a major pathway for removal of DNA base lesions and maintenance of genomic stability, which is essential in cancer prevention. DNA glycosylases recognize and remove specific lesions in the first step of BER. The existence of a number of these enzymes with overlapping substrate specificities has been thought to be the reason why single knock-out models of individual DNA glycosylases are not cancer prone. In this work we have characterized DNA glycosylases NEIL1 and NEIL2 (Neil1−/−/Neil2−/−) double and NEIL1, NEIL2 and NEIL3 (Neil1−/−/Neil2−/−/Neil3−/−) triple knock-out mouse models. Unexpectedly, our results show that these mice are not prone to cancer and have no elevated mutation frequencies under normal physiological conditions. Moreover, telomere length is not affected and there was no accumulation of oxidative DNA damage compared to wild-type mice. These results strengthen the hypothesis that the NEIL enzymes are not simply back-up enzymes for each other but enzymes that have distinct functions beyond canonical repair.

The putative drug efflux systems of the Bacillus cereus group.

The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70–80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current knowledge of the small molecule efflux pumps encoded by the B. cereus group and suggest the likely functions of numerous uncharacterised pumps.

Peroxisomal ATP-binding cassette transporters form mainly tetramers

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.

The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B

The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta, thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases.

Discovery of a Potent, Selective, and Cell-Active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6.

Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure-activity relationship (SAR) studies and explored three regions of this scaffold. The studies led to the discovery of a potent, selective, and cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and nonepigenetic targets. We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs, 17 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of PRMT4 and PRMT6 in health and disease.

Abstract 5021: Glutathione-S-transferases polymorphisms are associated with increased risk of relapse in pediatric patients with acute lymphoblastic leukemia

Abstract Current treatment protocols of the International Berlin-Frankfurt-Münster Study Group (BFM) for childhood Acute Lymphoblastic Leukemia (ALL) include the stratification of patients into groups of risk for disease relapse. This stratification is based on biochemical, molecular and cytogenetic parameters at diagnosis, and the early response to treatment determined by the minimal residual disease. ALL treatment protocols are continuously revised and improved, but some patients still recur and die of disease. The inclusion of genotype at ALL diagnosis as a genetic predictor of disease outcome is under constant study. However, results are inconclusive and seem to be population specific. Hence, we aimed to analyze the predictive value of germline polymorphisms for childhood ALL relapse in order to support their inclusion as additional tools in disease diagnosis and therapeutic protocols. We retrospectively recruited 140 Argentine patients diagnosed with de novo ALL (median age at diagnosis: 6.1 years old, range: 0-19). The protocol was approved by the Institutional Ethical Committee in compliance with the Ethical Principles enunciated by the Declaration of Helsinki. Genotypes were analyzed using PCR-RFLP (GSTP1 c.313A>G, MDR1 c.3435T>C, and MTHFR c.665C>T) and multiplex PCR (GSTT1 null, GSTM1 null). Relapse-free survival (RFS) was assessed using the Kaplan-Meier method and the risk for relapse was estimated using multivariate Cox proportional hazard models (median follow-up time: 52 months). Multivariate models included gender, risk group, treatment protocol, age at diagnosis and genotypes as covariates. Patients with the GSTP1 c.313GG genotype had worse RFS (p = 0.025) and a 2.6-fold increased risk for relapse (95%CI = 1.1-6.1, p = 0.030). In addition, since GSTs are enzymes that frequently participate in the same biological pathways with overlapping substrate specificity, we considered an additive score that captures information on the genotypes of GSTT1, GSTM1 and GSTP1. We found that patients with higher number of risk alleles genotype had statistically significant shorter RFS (p = 0.021) and over a 2.5-fold increased risk for relapse (95%CI = 1.1-5.6, p = 0.027). GST polymorphisms seemed to modify the risk of relapse and RFS of Argentine patients with childhood ALL. The inclusion of these genetic markers in ALL treatment protocols might improve risk stratification and reduce the number of relapses and deaths. Citation Format: María M. Abbate, Daiana B. Leonardi, Javier N. Brandani, María C. Riccheri, Geraldine Gueron, Graciela Alfonso, Elba S. Vazquez, Javier Cotignola. Glutathione-S-transferases polymorphisms are associated with increased risk of relapse in pediatric patients with acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5021.

Renal Transporter-Mediated Drug-Drug Interactions: Are They Clinically Relevant?

The kidney, through the distinct processes of passive glomerular filtration and active tubular secretion, plays an important role in the elimination of numerous endobiotics (eg, hormones, metabolites), toxins, nutrients, and drugs. Renal transport pathways mediating active tubular secretion and reabsorption in the proximal tubule are complex, involving apical and basolateral transporters acting in concert. Detailed studies of the molecular mechanisms of net active tubular secretion have established the involvement of multiple transporters with overlapping substrate specificity mediating competing secretion and reabsorption pathways. Although drug interactions arising from inhibition of renal transporters are rare relative to other mechanisms, they can involve commonly administered drugs (eg, cimetidine, metformin), may be underappreciated due to muted effects on plasma pharmacokinetics relative to tissue levels, can affect narrow-therapeutic-index medications (eg, antiarrhythmic, oncology medications), and may disproportionately affect sensitive populations where polypharmacy is common (eg, the elderly, diabetics). In particular, there is the potential for larger-magnitude interactions in subjects with reduced glomerular filtration rates due to the increased relative contribution of tubular secretion. The assessment of additional endpoints in drug-drug interaction studies including pharmacodynamics, positron emission tomography imaging, and metabolomics promises to expand our understanding of the clinical relevance of renal drug interactions.

Cysteinyl Leukotriene Receptor 1/2 Antagonists Nonselectively Modulate Organic Anion Transport by Multidrug Resistance Proteins (MRP1-4).

Active efflux of both drugs and organic anion metabolites is mediated by the multidrug resistance proteins (MRPs). MRP1 (ABCC1), MRP2 (ABCC2), MRP3 (ABCC3), and MRP4 (ABCC4) have partially overlapping substrate specificities and all transport 17β-estradiol 17-(β-d-glucuronide) (E217βG). The cysteinyl leukotriene receptor 1 (CysLT1R) antagonist MK-571 inhibits all four MRP homologs, but little is known about the modulatory effects of newer leukotriene modifiers (LTMs). Here we examined the effects of seven CysLT1R- and CysLT2R-selective LTMs on E217βG uptake into MRP1-4-enriched inside-out membrane vesicles. Their effects on uptake of an additional physiologic solute were also measured for MRP1 [leukotriene C4 (LTC4)] and MRP4 [prostaglandin E2 (PGE2)]. The two CysLT2R-selective LTMs studied were generally more potent inhibitors than CysLT1R-selective LTMs, but neither class of antagonists showed any MRP selectivity. For E217βG uptake, LTM IC50s ranged from 1.2 to 26.9 μM and were most comparable for MRP1 and MRP4. The LTM rank order inhibitory potencies for E217βG versus LTC4 uptake by MRP1, and E217βG versus PGE2 uptake by MRP4, were also similar. Three of four CysLT1R-selective LTMs also stimulated MRP2 (but not MRP3) transport and thus exerted a concentration-dependent biphasic effect on MRP2. The fourth CysLT1R antagonist, LY171883, only stimulated MRP2 (and MRP3) transport but none of the MRPs were stimulated by either CysLT2R-selective LTM. We conclude that, in contrast to their CysLTR selectivity, CysLTR antagonists show no MRP homolog selectivity, and data should be interpreted cautiously if obtained from LTMs in systems in which more than one MRP is present.

Computational modelling of placental amino acid transfer as an integrated system

Placental amino acid transfer is essential for fetal development and its impairment is associated with poor fetal growth. Amino acid transfer is mediated by a broad array of specific plasma membrane transporters with overlapping substrate specificity. However, it is not fully understood how these different transporters work together to mediate net flux across the placenta. Therefore the aim of this study was to develop a new computational model to describe how human placental amino acid transfer functions as an integrated system. Amino acid transfer from mother to fetus requires transport across the two plasma membranes of the placental syncytiotrophoblast, each of which contains a distinct complement of transporter proteins. A compartmental modelling approach was combined with a carrier based modelling framework to represent the kinetics of the individual accumulative, exchange and facilitative classes of transporters on each plasma membrane. The model successfully captured the principal features of transplacental transfer. Modelling results clearly demonstrate how modulating transporter activity and conditions such as phenylketonuria, can increase the transfer of certain groups of amino acids, but that this comes at the cost of decreasing the transfer of others, which has implications for developing clinical treatment options in the placenta and other transporting epithelia.

Spatio-Temporal Regulation of PKC Isoforms Imparts Signaling Specificity.

Nishizuka and colleagues discovered protein kinase C (PKC) as a calcium-dependent, lipid cofactor-sensitive protein kinase (1). Initially known as the only physiological effector for tumor-promoting phorbol esters, this ubiquitous enzyme eventually took center stage in cellular signaling. Part of the AGC kinase branch of the kinome, PKC is a family of serine/threonine kinases comprising 11 isoforms encoded by 9 genes and grouped into 4 classes – classical (cPKCs-α, βI, βII, γ), novel (nPKCs-δ, ϵ, η, θ), atypical (aPKCs-ζ, ι/λ), and PKCμ (a form between novel and atypical isoforms) (Box 1). Their considerable structural homology, overlapping substrate specificities, and biochemical properties indicated at least partial enzymatic redundancy and rendered the task of identifying isoform-specific functions challenging. The initial phase of PKC research correlated the unique structural features of the isoforms with their functions (Box 1) (2). But, with their structural overlaps and hugely varying functions in different models, specificity of the isoforms became a confounding puzzle demanding stringent isoform-specific regulation to avoid functional redundancy. To define this stringency, subsequent PKC research focused on the upstream and downstream regulatory mechanisms (3, 4), highlighting the importance of subcellular distribution as a function of time (5). As simultaneous activation of all PKC isoforms would be energetically and spatially conflicting for decoding the message received by the cell surface receptor, cell type, and stimulus-specific selective and sequential activation deemed justified (6). The recent phase of PKC research propounds a PKC-signaling module (7, 8) wherein an inter-isoform network regulates the PKC isoforms’ activity (Box 2; Figure ​Figure1B).1B). Here, we propose activator- and cofactor-specific sequential activation of PKC isoforms in a spatio-temporal model wherein selective subcellular compartmentalization quantitatively determines the isoform-specific effector functions. Box 1 Structural features of PKC isoforms defining functional specificity. PKC serve as a paradigm for the reversible regulation of membrane localization by the concerted action of two membrane-targeting modules. C1: a cysteine-rich region of approximately 50 residues present in all PKC isozymes. In cPKCs and nPKCs, it is present as a tandem repeat C1A and C1B. aPKCs contain a single copy of the domain termed atypical because it does not bind phorbol esters. The domain contains two pulled apart β-sheet forming the ligand-binding pocket. Two zinc atoms are coordinated by two histidines and six cysteines at opposite ends of the primary sequence stabilizing the domain. In aPKCs, one face of the ligand-binding pocket is compromised, so that the module cannot bind phorbol esters or DAG. Ligand binding dramatically alters the surface properties of the module (C1B). The ligand caps the hydrophilic ligand-binding pocket, so that the top-third of the C1 domain presents a continuous hydrophobic surface thus achieving membrane targeting by simply altering membrane properties. C2: an independent membrane-targeting module that binds calcium in cPKCs, but not in nPKCs. C2 domain is a β-strand rich globular domain with loops formed by sequences at opposite ends of the primary structure. Two topological variants exist – Type I for domains that follow the C1 domain (cPKCs) and Type II for domains that precede C1 (nPKCs). In the calcium-responsive C2 domains of cPKCs, the pocket is lined by multiple aspartic acid residues that coordinate two to three calcium ions that act as a bridge between the C2 domain and the phospholipid head groups of the membrane. CR, cysteine rich; PS, pseudosubstrate; DAG, diacylglycerol; PMA, phorbol-12-myristate 13-acetate; PB1, Phox–Bem 1; C, constant regions; V, variable regions.