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This letter proposes an ultrabroadband absorber (UBA) based on the layered inkjet-printing resistive film (IPRF) composed of three lossy layers. The equivalent circuit model of the UBA is put forward to analyze the impedance matching with air under broadband conditions. The prototype was fabricated using IPRF as the lossy layers and the polymethacrylimide (PMI) foam as the dielectric layers. The simulated and measured results show that the ultrabroadband 0 dB absorption performance covers 2–23 GHz with the fractional bandwidth of 168% and maintains stable absorbing performance under the incidence angle of 45°. The total thickness of the proposed design is only 0.1λL at the lowest operating frequency. Experimental validation of the fabricated prototype has also confirmed the potential using of IPRF-based broadband absorbers in several applications.

Scaffold hopping and structure-based drug design were employed to identify substituted 4-aminoquinolines and 4-aminonaphthyridines as potent, small molecule inhibitors of tumor necrosis factor alpha (TNFα). Structure–activity relationships in both the quinoline and naphthyridine series leading to the identification of compound 42with excellent potency and pharmacokinetic profile are discussed. X-ray co-crystal structure analysis and ultracentrifugation experiments clearly demonstrate that these inhibitors distort the TNFα trimer upon binding, leading to aberrant signaling when the trimer binds to TNF receptor 1 (TNFR1). Pharmacokinetic–pharmacodynamic activity of compound 42in a TNF-induced IL-6 mouse model and in vivo activity in a collagen antibody-induced arthritis model, where it showed biologic-like in vivo efficacy, will be discussed.

The NaV1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaVAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of NaV1.7. Importantly, this chimera, DII S1–S4, forms functional sodium channels and is potently inhibited by the NaV1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [125I]ProTx-II binding and surface plasmon resonance that the purified DII S1–S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1–S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a NaV1.7-NaVAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel NaV1.7 inhibitors and for structural studies to understand the toxin-channel interaction.

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.

While several farnesoid X receptor (FXR) agonists under clinical investigation for the treatment of nonalcoholic steatohepatitis (NASH) have shown beneficial effects, adverse effects such as pruritus and elevation of plasma lipids have limited their clinical efficacy and approvability. Herein, we report the discovery and preclinical evaluation of compound 32(BMS-986339), a nonbile acid FXR agonist with a pharmacologically distinct profile relative to our previously reported agonist BMS-986318. Compound 32exhibited potent in vitro and in vivo activation of FXR, albeit with a context-dependent profile that resulted in tissue-selective effects in vivo. To our knowledge, this is the first report that demonstrates differential induction of Fgf15in the liver and ileum by FXR agonists in vivo. Compound 32demonstrated robust antifibrotic efficacy despite reduced activation of certain genes in the liver, suggesting that the additional pharmacology of BMS-986318 does not further benefit efficacy, possibly presenting an opportunity for reduced adverse effects. Further evaluation in humans is warranted to validate this hypothesis.

Multidrug resistance proteins (MRPs) are members of the "C" branch of the ATP-binding cassette transporter superfamily. Human MRP1 transports a wide range of natural product drugs and structurally diverse conjugated and unconjugated organic anions. Its closest relative is MRP3. Despite their structural similarity, the homologs differ substantially in their substrate specificity. It is noteworthy that MRP1 transports glutathione (GSH) and GSH conjugates and displays GSH-stimulated transport of a number of unconjugated and conjugated compounds. In contrast, MRP3 does not transport GSH and is a poor transporter of GSH conjugates. However, both proteins transport glucuronide conjugates, such as 17β-estradiol 17-(β-D-glucuronide). We have constructed a series of MRP1/MRP3 hybrids and used them to identify a region of MRP1 that is critical for binding and transport of GSH conjugates such as leukotriene C4(LTC4). Substitution of this region encompassing transmembrane helices 8 and 9 and portions of cytoplasmic loops 4 and 5 of MRP1 with the equivalent region of MRP3 eliminated LTC4transport. Transport of other substrates was either unaffected or enhanced. We identified three residues in this region: Tyr440, Ile441, and Met443, mutation of which differentially affected transport. It is noteworthy that substitution of Tyr440with Phe, as found in MRP3, reduced LTC4and GSH-stimulated estrone-3-sulfate transport without affecting transport of other substrates tested. The mutation increased the Kmfor LTC45-fold and substantially reduced photolabeling of MRP1 by both [3H]LTC4and the GSH derivative, azidophenacyl-[35S]GSH. These results suggest that Tyr440makes a major contribution to recognition of GSH and the GSH moiety of conjugates such as LTC4.

Multidrug resistance protein 1 (MRP1) is a member of the "C" branch of the ATP-binding cassette transporter superfamily. The NH2-proximal nucleotide-binding domain (NBD1) of MRP1 differs functionally from its COOH-proximal domain (NBD2). NBD1 displays intrinsic high-affinity ATP binding and little ATPase activity. In contrast, ATP binding to NBD2 is strongly dependent on nucleotide binding by NBD1, and NBD2 is more hydrolytically active. We have demonstrated that occupancy of NBD2 by ATP or ADP markedly decreased substrate binding by MRP1. We have further explored the relationship between nucleotide and substrate binding by examining the effects of various ATP analogs and ADP trapping, as well as mutations in conserved functional elements in the NBDs, on the ability of MRP1 to bind the photoactivatable, high-affinity substrate cysteinyl leukotriene C4 (LTC4). Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in the formation of a low-affinity conformation of the protein. However, nonhydrolyzable ATP analogs (β,γ-imidoadenosine 5'-triphosphate and adenylylmethylene diphosphonate) failed to substitute for ATP or adenosine 5'-O-(thiotriphosphate) (ATPγS) in decreasing LTC4 photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding abrogated the formation of a low-affinity substrate binding state in the presence of ATP or ATPγS. We suggest that the effect of these mutations, and possibly the failure of some ATP analogs to decrease LTC4 binding, may be attributable to an inability to elicit a conformational change in the NBDs that involves interactions between the signature sequence and the γ-phosphate of the bound nucleotide.

Multidrug resistance protein 1 (MRP1) is capable of actively transporting a wide range of conjugated and unconjugated organic anions. The protein can also transport additional conjugated and unconjugated compounds in a GSH- or S-methyl GSH-stimulated manner. How MRP1 binds and transports such structurally diverse substrates is not known. We have used [3H]leukotriene C4(LTC4), a high affinity glutathione-conjugated physiological substrate, to photolabel intact MRP1, as well as fragments of the protein expressed in insect cells. These studies revealed that: (i) LTC4labels sites in the NH2- and COOH-proximal halves of MRP1, (ii) labeling of the NH2-half of MRP1 is localized to a region encompassing membrane-spanning domain (MSD) 2 and nucleotide binding domain (NBD) 1, (iii) labeling of this region is dependent on the presence of all or part of the cytoplasmic loop (CL3) linking MSD1 and MSD2, but not on the presence of MSD1, (iv) labeling of the NH2-proximal site is preferentially inhibited byS-methyl GSH, (v) labeling of the COOH-proximal half of the protein occurs in a region encompassing transmembrane helices 14–17 and appears not to require NBD2 or the cytoplasmic COOH-terminal region of the protein, (vi) labeling of intact MRP1 by LTC4is strongly attenuated in the presence of ATP and vanadate, and this decrease in labeling is attributable to a marked reduction in LTC4binding to the NH2-proximal site, and (vii) the attenuation of LTC4binding to the NH2-proximal site is a consequence of ATP hydrolysis and trapping of Vi-ADP exclusively at NBD2. These data suggest that MRP1-mediated transport involves a conformational change, driven by ATP hydrolysis at NBD2, that alters the affinity with which LTC4binds to one of two sites composed, at least in part, of elements in the NH2-proximal half of the protein.

Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics. The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates. It is not known whether the two NBDs of MRP1 are functionally equivalent. To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-[32P]ATP and to trap 8-azido-[32P]ADP in the presence of orthovanadate. We found that NBD1 was preferentially labeled with 8-azido-[32P]ATP, while trapping of 8-azido-[32P]ADP occurred predominantly at NBD2. Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH2-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide. Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2. Both mutations decreased leukotriene C4(LTC4) transport by approximately 70%. Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC4transport activity without affecting labeling of NBD1 with 8-azido-[32P]ATP.

Multidrug resistance in tumor cells is often accompanied by overexpression of multidrug resistance protein (MRP), a 190-kDa transmembrane protein that belongs to the ATP-binding cassette superfamily of transport proteins. MRP mediates ATP-dependent transport of a variety of conjugated organic anions and can also transport several unmodified xenobiotics in a glutathione-dependent manner. To facilitate structure-function studies of MRP, we have generated a panel of MRP-specific monoclonal antibodies (mAbs). Four of these mAbs, QCRL-2, -3, -4, and -6, bind intracellular conformation-dependent epitopes, and we have shown that they can inhibit the transport of several MRP substrates. Binding competition and immunoprecipitation assays indicated that mAbs QCRL-4 and -6 probably recognize the same detergent-sensitive epitope in MRP, whereas mAbs QCRL-2, -3, and -4 each bind distinct, non-overlapping epitopes. Fab fragments inhibit transport as effectively as the intact mAbs, suggesting that inhibition results from direct interactions of the mAbs with MRP. Immunodot blot and immunoprecipitation analyses revealed that the minimal regions of MRP sufficient for full reactivity of mAbs QCRL-2 and -3 are amino acids 617–858 and 617–932, respectively, which encompass the NH2-proximal nucleotide-binding domain (NBD). In contrast, the epitope bound by mAb QCRL-4 localized to amino acids 1294–1531, a region that contains the COOH-proximal NBD. However, none of the mAbs inhibited photolabeling of intact MRP with 8-azido-[α-32P]ATP. This suggests that rather than preventing nucleotide binding, the mAbs inhibit transport by interfering with substrate binding or by trapping MRP in a conformation that does not allow transport to occur. Our results also demonstrate for the first time that the NBDs of MRP can be expressed as soluble polypeptides that retain a native conformation.

Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells. The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains. The role(s) of the third MSD of MRP and its related transporters is not known. To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C4(LTC4). We found that elimination of the entire NH2-terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/MRP2), had little effect on protein accumulation in the membrane. However, all three modifications decreased LTC4transport activity by at least 90%. Transport activity could be reconstituted by co-expression of the NH2-terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH2-terminal MSD and the cytoplasmic region linking it to the next MSD. In contrast, a major part of the cytoplasmic region linking the NH2-proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC4.

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs is unknown. To facilitate studies on the structure and function of MRP, we have determined whether a baculovirus expression system can be used to produce active protein. Full-length MRP as well as molecules corresponding to either the NH2- or COOH-proximal halves of the protein were expressed individually and in combination in Spodoptera frugiperdaSf21 cells. High levels of intact and half-length proteins were detected in membrane vesicles from infected cells. Although underglycosylated, the full-length protein transported leukotriene C4(LTC4) with kinetic parameters very similar to those of MRP produced in transfected HeLa cells. Neither half-molecule was able to transport LTC4. However, a functional transporter with characteristics similar to those of intact protein could be reconstituted when both half-molecules were co-expressed. Transport of LTC4by Sf21 membrane vesicles containing either intact or reconstituted MRP was competitively inhibited by both S-decylglutathione and 17β-estradiol 17-(β-D-glucuronide), with Kivalues similar to those reported previously for MRP expressed in HeLa cells (Loe, D. W., Almquist, K. C., Deeley, R. G., and Cole, S. P. C. (1996) J. Biol. Chem.271, 9675-9682; Loe, D. W., Almquist, K. C., Cole, S. P. C., and Deeley, R. G. (1996) J. Biol. Chem.271, 9683-9689). These studies demonstrate that human MRP produced in insect cells can function as an active transporter of LTC4and that the NH2- and COOH-proximal halves of the protein can assemble efficiently to form a transporter with functional characteristics similar to those of the intact protein.