Multidrug Resistance Protein 1 (MRP1) 1 is a member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that has been shown to confer resistance to a variety of natural product type drugs (1-6). The drug resistance phenotype conferred by MRP1 is similar to that resulting from overexpression of P-glycoprotein (P-gp) (reviewed in Refs. 7-9) and is typically associated with an ATP-dependent decrease in drug accumulation and an increase in drug efflux (4, 6). Although both ABC proteins can function as energy-dependent efflux pumps for a range of natural product type drugs, there is very limited primary structure similarity between them, and phylogenetic analyses suggest that they evolved from different ancestral proteins. There is also considerable evidence that the mechanisms by which MRP1 and P-gp transport drugs are different (reviewed in Ref. 8).In addition to its ability to confer multidrug resistance, MRP1, unlike P-gp, has been shown by in vitro studies using inside-out membrane vesicles to transport a structurally diverse array of organic, anionic conjugates (reviewed in Ref. 9). These include GSH-, glucuronide-, and sulfate-conjugated aliphatic, prostanoid, and heterocyclic compounds. The two highest affinity substrates identified to date are the proinflammatory cysteinyl leukotriene C 4 (LTC 4 ) (10 -12) and the GSH-

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MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.ATP-binding cassette (ABC) 1 transporters are ubiquitous transmembrane proteins that couple ATP hydrolysis to the energy-dependent transport of a wide variety of endogenous and exogenous molecules across biological membranes. Multidrug resistance protein (MRP) 1 (ABCC1) belongs to the "C" subfamily of the ABC superfamily and was discovered by virtue of its ability to cause multidrug resistance when overexpressed in a human small cell lung cancer cell line (1, 2). The MRP1 multidrug resistance phenotype is similar to that resulting from overexpression of P-glycoprotein (P-GP), and involves resistance to many relatively hydrophobic, natural product type, cytotoxic agents. However, unlike P-GP, MRP1 can also transport various structurally unrelated organic anionic conjugates, including: glutathione, glucuronide, and sulfate conjugates, such as the potent mediator of inflammation, cysteinyl leukotriene LTC 4 , the cholestatic glucuronide-conjugated estrogen E 2 17␤G, the sulfate conjugate estrone 3-sulfate, and the glutathione epoxide conjugate of the highly mutagenic aflatoxin B1 (3-10).Typically, the functional form of ABC proteins consists of two hydrophilic nucleotide-binding domains (NBDs) located at the cytoplasmic surface of the membrane and two hydrophobic transmembrane spanning domains (MSD) that are thought to for...

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.

Proprotein convertase subtilisin kexin-9 (PCSK9) is an important pharmacological target for decreasing low-density lipoprotein (LDL) in cardiovascular disease, although seemingly inaccessible to small molecule approaches. Compared with therapeutic IgG antibodies currently in development, targeting circulating PCSK9 with smaller molecular scaffolds could offer different profiles and reduced dose burdens. This inspired genesis of PCSK9-binding Adnectins, a protein family derived from human fibronectin-10th-type III-domain and engineered for high-affinity target binding. BMS-962476, an ∼11-kDa polypeptide conjugated to polyethylene glycol to enhance pharmacokinetics, binds with subnanomolar affinity to human. The X-ray cocrystal structure of PCSK9 with a progenitor Adnectin shows ∼910 Å 2 of PCSK9 surface covered next to the LDL receptor binding site, largely by residues of a single loop of the Adnectin. In hypercholesterolemic, overexpressing human PCSK9 transgenic mice, BMS-962476 rapidly lowered cholesterol and free PCSK9 levels. In genomic transgenic mice, BMS-962476 potently reduced free human PCSK9 (ED 50 ∼0.01 mg/kg) followed by ∼2-fold increases in total PCSK9 before return to baseline. Treatment of cynomolgus monkeys with BMS-962476 rapidly suppressed free PCSK9 .99% and LDL-cholesterol ∼55% with subsequent 6-fold increase in total PCSK9, suggesting reduced clearance of circulating complex. Liver sterol response genes were consequently downregulated, following which LDL and total PCSK9 returned to baseline. These studies highlight the rapid dynamics of PCSK9 control over LDL and liver cholesterol metabolism and characterize BMS-962476 as a potent and efficacious PCSK9 inhibitor.