Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. . NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.

Nuclear magnetic resonance (NMR) is arguably the most direct methodology for characterizing the higher-order structure of proteins in solution. Structural characterization of proteins by NMR typically utilizes heteronuclear experiments. However, for formulated monoclonal antibody (mAb) therapeutics, the use of these approaches is not currently tenable due to the requirements of isotope labeling, the large size of the proteins, and the restraints imposed by various formulations. Here, we present a new strategy to characterize formulated mAbs using (1)H NMR. This method, based on the pulsed field gradient stimulated echo (PGSTE) experiment, facilitates the use of (1)H NMR to generate highly resolved spectra of intact mAbs in their formulation buffers. This method of data acquisition, along with postacquisition signal processing, allows the generation of structural and hydrodynamic profiles of antibodies. We demonstrate how variation of the PGSTE pulse sequence parameters allows proton relaxation rates and relative diffusion coefficients to be obtained in a simple fashion. This new methodology can be used as a robust way to compare and characterize mAb therapeutics.

Restoration of p53 function through the disruption of the MDM2-p53 protein complex is a promising strategy for the treatment of various types of cancer. Here, we present kinetic, thermodynamic, and structural rationale for the remarkable potency of a new class of MDM2 inhibitors, the piperidinones. While these compounds bind to the same site as previously reported for small molecule inhibitors, such as the Nutlins, data presented here demonstrate that the piperidinones also engage the N-terminal region (residues 10-16) of human MDM2, in particular, Val14 and Thr16. This portion of MDM2 is unstructured in both the apo form of the protein and in MDM2 complexes with p53 or Nutlin, but adopts a novel β-strand structure when complexed with the piperidinones. The ordering of the N-terminus upon binding of the piperidinones extends the current model of MDM2-p53 interaction and provides a new route to rational design of superior inhibitors.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

The CbpA peptide-L460D pneumolysoid fusion protein was broadly protective against pneumococcal infection, with the potential for additional protection against other meningeal pathogens.

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a proteinprotein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only ϳ2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication. Hepatitis C virus (HCV),5 a member of the family Flaviviridae, is a leading cause of liver cirrhosis and hepatocellular carcinoma and has infected more than 170 million people worldwide (1-3). Standard treatment with interferon-␣ and ribavirin is frequently ineffective or toxic, and no vaccine for HCV is currently available (4). Identification of a novel viral target for therapeutic intervention could lead to the development of a more effective treatment.The HCV genome is a 9.6-kb positive, single-stranded RNA that supports translation of a 3000-amino acid polyprotein, which is subsequently cleaved to produce both structural (C, E1, and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) viral proteins (5). Two viral proteases are encoded by the NS2 and NS3 genes (6, 7). In addition to its protease domain, the NS3 protein also contains a DEXH/ D-box helicase domain (8). NS4A encodes a peptide co-factor for NS3 protease (9), and NS5B encodes an RNA-dependent RNA polymerase (10). The functions of NS4B and NS5A have not been as well characterized, but NS5A is involved in viral interferon sensitivity (11) and in adaptation of subgenomic replicons for growth in cultured hepatoma cells (12).One obstacle to studying viral protein interactions is that HCV cannot be maintained efficiently in cell culture, although recent progress may overcome this difficulty (13). In order to circumvent this problem, selectable replicons carrying the neomycin phosphotransferase gene in add...

Evidence has been presented (Kukar et al. 2008 Nature 453, 925-929) that certain γ-secretase modulators (GSMs) target the 99 residue C-terminal domain (C99) of the amyloid precursor protein, a substrate of γ-secretase, but not the protease complex itself. Here, NMR results demonstrate a lack of specific binding of these GSMs to monodisperse C99 in LMPG micelles. In addition, results indicate that C99 was likely to have been aggregated in some of the key experiments of the previous work, and that binding of GSMs to these C99 aggregates is also of a non-specific nature.Among the therapeutic targets for Alzheimer's disease (AD), the amyloid pathway has long been paramount (1). Familial early-onset AD (FAD) is associated with autosomal dominant mutations in the amyloid precursor protein (APP) and in the catalytic subunits (presenilin 1 and presenilin 2) of the intramembrane protease that processes it, γ-secretase (2). According to the amyloid hypothesis, oligomeric forms of Aβ are the principal agents underlying disease pathogenesis (1). The Aβ peptide is generated by proteolysis of APP. Cleavage of APP by β-secretase yields C99 2 , which is then heterogeneously processed by γ-secretase to generate Aβ species with a variety of lengths, principally Aβ40 (3). Familial AD is associated with an increase in the Aβ42/Aβ40 ratio, with Aβ42 being the primary species deposited in the brain parenchyma of most individuals with AD (4). Because Aβ is thought to be central to the pathogenesis of AD, inhibiting its production is a potential therapeutic strategy (1). Although significant progress has been made in the identification and development of potent γ-secretase inhibitors, their clinical application has been limited by significant toxicities resulting from interference with processing of other γ-secretase substrates, particularly Notch (5). Indeed, γ-secretase is a highly promiscuous protease with more than sixty identified targets (6).The discovery that a subset of non-steroidal anti-inflammatory drugs could selectively reduce Aβ42 production without abrogating Notch cleavage suggested an alternative therapeutic strategy for AD (7). The Aβ42-lowering activity of these γ-secretase modulators (GSMs) was recapitulated in cell-free assays of γ-secretase activity. Several groups have produced data suggesting that GSMs interact allosterically with presenilin, thereby modifying the enzyme's conformation (8-10). Moreover, GSMs were observed to influence the cleavage of an unrelated substrate by signal peptide peptidase -an enzyme with homology to the presenilin subunit of γ-secretase, suggesting that the modulators interact with the enzyme rather than substrate † This work was supported by grants from the US NIH (PO1 GM080513, to CRS) and from the Alzheimer's Association (IIRG-07-59379, to CRS). A recent report from the Golde laboratory, however, postulates that GSMs specifically target APP and its C-terminal derivatives, providing an alternative explanation to the apparent specificity that GSMs exert on cleavage of C99 (1...