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Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 tha

Optical fiber gratings (OFGs), especially long-period gratings (LPGs) and etched or tilted fiber Bragg gratings (FBGs), are playing an increasing role in the chemical and biochemical sensing based on the measurement of a surface refractive index (RI) change through a label-free configuration. In these devices, the electric field evanescent wave at the fiber/surrounding medium interface changes its optical properties (i.e. intensity and wavelength) as a result of the RI variation due to the interaction between a biological recognition layer deposited over the fiber and the analyte under investigation. The use of OFG-based technology platforms takes the advantages of optical fiber peculiarities, which are hardly offered by the other sensing systems, such as compactness, lightness, high compatibility with optoelectronic devices (both sources and detectors), and multiplexing and remote measurement capability as the signal is spectrally modulated. During the last decade, the growing request in practical applications pushed the technology behind the OFG-based sensors over its limits by means of the deposition of thin film overlays, nanocoatings, and nanostructures, in general. Here, we review efforts toward utilizing these nanomaterials as coatings for high-performance and low-detection limit devices. Moreover, we review the recent development in OFG-based biosensing and identify some of the key challenges for practical applications. While high-performance metrics are starting to be achieved experimentally, there are still open questions pertaining to an effective and reliable detection of small molecules, possibly up to single molecule, sensing in vivo and multi-target detection using OFG-based technology platforms.

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.

Background: After performing a fragment based screen the resulting hits need to be prioritized for follow-up structure elucidation and chemistry. This paper describes a new similarity metric, Atom-Atom-Path (AAP) similarity that is used in conjunction with the Directed Sphere Exclusion (DISE) clustering method to effectively organize and prioritize the fragment hits. The AAP similarity rewards common substructures and recognizes minimal structure differences. The DISE method is order-dependent and can be used to enrich fragments with properties of interest in the first clusters., Results: The merit of the software is demonstrated by its application to the MAP4K4 fragment screening hits using ligand efficiency (LE) as quality measure. The first clusters contain the hits with the highest LE. The clustering results can be easily visualized in a LE-over-clusters scatterplot with points colored by the members' similarity to the corresponding cluster seed. The scatterplot enables the extraction of preliminary SAR., Conclusions: The detailed structure differentiation of the AAP similarity metric is ideal for fragment-sized molecules. The order-dependent nature of the DISE clustering method results in clusters ordered by a property of interest to the teams. The combination of both allows for efficient prioritization of fragment hit for follow-ups. Graphical abstractAAP similarity computation and DISE clustering visualization.

Cellular inhibitor of apoptosis 1 (cIAP1) is a ubiquitin ligase with critical roles in the control of programmed cell death and NF-κB signaling. Under normal conditions, the protein exists as an autoinhibited monomer, but proapoptotic signals lead to its dimerization, activation and proteasomal degradation. This view of cIAP1 as a binary switch has been informed by static structural studies that cannot access the protein's dynamics. Here, we use NMR spectroscopy to study micro- and millisecond motions of specific domain interfaces in human cIAP1 and use time-resolved small-angle X-ray scattering to observe the global conformational changes necessary for activation. Although motions within each interface of the 'closed' monomer are insufficient to activate cIAP1, they enable associations with catalytic partners and activation factors. We propose that these internal motions facilitate rapid peptide-induced opening and dimerization of cIAP1, which undergoes a dramatic spring-loaded structural transition.

MAP4K4 has been shown to regulate key cellular processes that are tied to disease pathogenesis. In an effort to generate small molecule MAP4K4 inhibitors, a fragment-based screen was carried out and a pyrrolotriazine fragment with excellent ligand efficiency was identified. Further modification of this fragment guided by X-ray crystal structures and molecular modeling led to the discovery of a series of promising compounds with good structural diversity and physicochemical properties. These compounds exhibited single digit nanomolar potency and compounds 35 and 44 achieved good in vivo exposure., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.

Potent, trans-2-(pyridin-3-yl)cyclopropanecarboxamide-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using fragment-based screening and structure-based design techniques. Multiple crystal structures were obtained of initial fragment leads, and this structural information was utilized to improve the biochemical and cell-based potency of the associated molecules. Many of the optimized compounds exhibited nanomolar antiproliferative activities against human tumor lines in in vitro cell culture experiments. In a key example, a fragment lead (13, KD = 51 μM) was elaborated into a potent NAMPT inhibitor (39, NAMPT IC50 = 0.0051 μM, A2780 cell culture IC50 = 0.000 49 μM) which demonstrated encouraging in vivo efficacy in an HT-1080 mouse xenograft tumor model.

The fragment-based identification of two novel and potent biochemical inhibitors of the nicotinamide phosphoribosyltransferase (NAMPT) enzyme is described. These compounds (51 and 63) incorporate an amide moiety derived from 3-aminopyridine, and are thus structurally distinct from other known anti-NAMPT agents. Each exhibits potent inhibition of NAMPT biochemical activity (IC50=19 and 15 nM, respectively) as well as robust antiproliferative properties in A2780 cell culture experiments (IC50=121 and 99 nM, respectively). However, additional biological studies indicate that only inhibitor 51 exerts its A2780 cell culture effects via a NAMPT-mediated mechanism. The crystal structures of both 51 and 63 in complex with NAMPT are also independently described., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure-activity relationships., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The use of fragments with low binding affinity for their targets as starting points has received much attention recently. Screening of fragment libraries has been the most common method to find attractive starting points. Herein, we describe a unique, alternative approach to generating fragment leads. A binding model was developed and a set of guidelines were then selected to use this model to design fragments, enabling our discovery of a novel fragment with high LE.

The Ras gene is frequently mutated in cancer, and mutant Ras drives tumorigenesis. Although Ras is a central oncogene, small molecules that bind to Ras in a well-defined manner and exert inhibitory effects have not been uncovered to date. Through an NMR-based fragment screen, we identified a group of small molecules that all bind to a common site on Ras. High-resolution cocrystal structures delineated a unique ligand-binding pocket on the Ras protein that is adjacent to the switch I/II regions and can be expanded upon compound binding. Structure analysis predicts that compound-binding interferes with the Ras/SOS interactions. Indeed, selected compounds inhibit SOS-mediated nucleotide exchange and prevent Ras activation by blocking the formation of intermediates of the exchange reaction. The discovery of a small-molecule binding pocket on Ras with functional significance provides a new direction in the search of therapeutically effective inhibitors of the Ras oncoprotein.

Inhibition of caspase-6 is a potential therapeutic strategy for some neurodegenerative diseases, but it has been difficult to develop selective inhibitors against caspases. We report the discovery and characterization of a potent inhibitor of caspase-6 that acts by an uncompetitive binding mode that is an unprecedented mechanism of inhibition against this target class. Biochemical assays demonstrate that, while exquisitely selective for caspase-6 over caspase-3 and -7, the compound's inhibitory activity is also dependent on the amino acid sequence and P1' character of the peptide substrate. The crystal structure of the ternary complex of caspase-6, substrate-mimetic and an 11 nM inhibitor reveals the molecular basis of inhibition. The general strategy to develop uncompetitive inhibitors together with the unique mechanism described herein provides a rationale for engineering caspase selectivity.

Inhibitor of apoptosis (IAP) proteins are negative regulators of cell death. IAP family members contain RING domains that impart E3 ubiquitin ligase activity. Binding of endogenous or small-molecule antagonists to select baculovirus IAP repeat (BIR) domains within cellular IAP (cIAP) proteins promotes autoubiquitination and proteasomal degradation and so releases inhibition of apoptosis mediated by cIAP. Although the molecular details of antagonist-BIR domain interactions are well understood, it is not clear how this binding event influences the activity of the RING domain. Here biochemical and structural studies reveal that the unliganded, multidomain cIAP1 sequesters the RING domain within a compact, monomeric structure that prevents RING dimerization. Antagonist binding induces conformational rearrangements that enable RING dimerization and formation of the active E3 ligase.

Vismodegib (GDC-0449) is is an orally available selective Hedgehog pathway inhibitor in development for cancer treatment. The drug is ≥95% protein bound in plasma at clinically relevant concentrations and has an approximately 200-fold longer single dose half-life in humans than rats. We have identified a strong linear relationship between plasma drug concentrations and α-1-acid glycoprotein (AAG) in a phase I study. Biophysical and cellular techniques have been used to reveal that vismodegib strongly binds to human AAG (K(D) = 13 μM) and binds albumin with lower affinity (K(D) = 120 μM). Additionally, binding to rat AAG is reduced ∼20-fold relative to human, whereas the binding affinity to rat and human albumin was similar. Molecular docking studies reveal the reason for the signficiant species dependence on binding. These data highlight the utility of biophysical techniques in creating a comprehensive picture of protein binding across species.

The detection and characterization of fragment binding requires the use of technologies with extreme sensitivity to observe the binding interactions of low-affinity and low-molecular weight compounds to proteins. A number of methods have emerged capable of providing fragment hits to project teams including, but certainly not limited to, NMR, X-ray crystallography, and surface plasmon resonance (SPR). SPR-based biosensors are sufficiently sensitive and high throughput to provide complete fragment screens on libraries of several thousand compounds in just a few weeks per target. Biosensors provide quantitative binding information for ranking fragments by affinity and ligand efficiency and can support ongoing quantitative structure-activity efforts during fragment hit-to-lead development. The combination of speed and binding quantitation makes SPR a valuable technology in pharmaceutical fragment-based drug discovery and development. Successful implementation of SPR biosensors in fragment efforts requires specialized methods for instrument preparation, assay development, primary compound handling, primary screening, confirmation testing, and data analysis. In this chapter, each of these topics is discussed in detail with general best practices for maintaining the highest throughput while maximizing data quality., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

The binding affinity of four palm and thumb site representative non-nucleoside inhibitors (NNIs) of HCV polymerase NS5B to wild-type and resistant NS5B polymerase proteins was determined, and the influence of RNA binding on NNI binding affinity was investigated. NNIs with high binding affinity potently inhibited HCV RNA polymerase activity and replicon replication. Among the compounds tested, HCV-796 showed slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 +/- 2 nm. Slow binding kinetics of HCV-796 was driven by slow dissociation from NS5B with a k(off) of 4.9 +/- 0.5 x 10(-4) s(-1). NS5B bound a long, 378-nucleotide HCV RNA oligonucleotide with high affinity (Kd = 6.9 +/- 0.3 nm), whereas the binding affinity was significantly lower for a short, 21-nucleotide RNA (Kd = 155.1 +/- 16.2 nm). The formation of the NS5B-HCV RNA complex did not affect the slow binding kinetics profile and only slightly reduced NS5B binding affinity of HCV-796. The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5B polymerase activity and replicon assays. Co-crystal structures of NS5B-Con1 and NS5B-BK with HCV-796 revealed a deep hydrophobic binding pocket at the palm region of NS5B. HCV-796 interaction with the induced binding pocket on NS5B is consistent with slow binding kinetics and loss of binding affinity with mutations at amino acid position 316.

Fragment screens for new ligands have had wide success, notwithstanding their constraint to libraries of 1,000-10,000 molecules. Larger libraries would be addressable were molecular docking reliable for fragment screens, but this has not been widely accepted. To investigate docking's ability to prioritize fragments, a library of >137,000 such molecules were docked against the structure of beta-lactamase. Forty-eight fragments highly ranked by docking were acquired and tested; 23 had K(i) values ranging from 0.7 to 9.2 mM. X-ray crystal structures of the enzyme-bound complexes were determined for 8 of the fragments. For 4, the correspondence between the predicted and experimental structures was high (RMSD between 1.2 and 1.4 A), whereas for another 2, the fidelity was lower but retained most key interactions (RMSD 2.4-2.6 A). Two of the 8 fragments adopted very different poses in the active site owing to enzyme conformational changes. The 48% hit rate of the fragment docking compares very favorably with "lead-like" docking and high-throughput screening against the same enzyme. To understand this, we investigated the occurrence of the fragment scaffolds among larger, lead-like molecules. Approximately 1% of commercially available fragments contain these inhibitors whereas only 10(-7)% of lead-like molecules do. This suggests that many more chemotypes and combinations of chemotypes are present among fragments than are available among lead-like molecules, contributing to the higher hit rates. The ability of docking to prioritize these fragments suggests that the technique can be used to exploit the better chemotype coverage that exists at the fragment level.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Biacore T100 technology was used in conjunction with a van't Hoff analysis to characterize the thermodynamic binding parameters of 85 small-molecule inhibitors of adenosine triphosphate (ATP) binding to p38 mitogen-activated protein (MAP) kinase. The compounds were selected from a large panel of azaindole and pyrazole derivatives for which IC(50) data exist. We showed a strong relationship between the K(D) and IC(50) of a compound, but only a modest relationship between k(off) and IC(50) was detected and an apparent relationship between a compound's k(on) and its IC(50) could not be discerned. Similarly, a correlation between a compound's IC(50) and its thermodynamic parameters DeltaH degrees and DeltaS degrees could not be established. The lack of a predominant kinetic or thermodynamic signature associated with the inhibitory potential of these compounds demonstrates that there exists, even within a single well-defined system, a library of kinetic routes or, alternatively, a library of initial and final enthalpic and entropic states from which to effect inhibition. As a complement to these studies, selected double mutant thermodynamic cycles were performed to probe the energetic coupling, if any, between common sites of fluorination in both the azaindole and pyrazole classes and two different substituents. Although both cycles indicated negligible coupling free energies, both revealed significant coupling enthalpies, an observation made in other similarly dissected systems. The possible significance and caveats associated with these findings along with the advantages of using Biacore technology to derive thermodynamic parameters in drug discovery efforts are discussed.

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340-356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an "OR-gate" type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.

Novel non-nucleoside inhibitors of HIV-RT that contain pyridazinone isosteres were prepared, and a series of triazolinones were found to be potent inhibitors of HIV replication. These compounds were active against several NNRTI-resistant virus strains. Pharmacokinetic studies indicated that inhibitor 7e has good bioavailability in rats. Several fragments of inhibitor 7c were prepared, and the binding of these compounds to HIV-RT was analyzed by surface plasmon resonance spectroscopy.

Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with approximately 1-10mM binding affinity (K(D)) were iteratively optimized to give leads with approximately 200nM biochemical activity and low microM cellular activity in a Replicon assay.

Hemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe-/- mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1.

Promiscuous binders achieve enzyme inhibition using a nonspecific aggregation-type binding mechanism to proteins. These compounds are a source of false-positive hits in biochemical inhibition assays and should be removed from screening hit lists because they are not good candidates to initiate medicinal chemistry programs. We introduce a robust approach to identify these molecules early in the lead generation process using real time surface plasmon resonance based biosensors to observe the behavior of the binding interactions between promiscuous compounds and proteins. Furthermore, the time resolution of the assay reveals a number of distinct mechanisms that promiscuous compounds employ to inhibit enzyme function and indicate that the type of mechanism can vary depending on the protein target. A classification scheme for these compounds is presented that can be used to rapidly characterize the hits from high-throughput screens and eliminate compounds with a nonspecific mechanism of inhibition.

Calmodulin (CaM) trapping by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a phenomenon whereby the affinity of CaM for CaMKII increases >1000-fold following CaMKII autophosphorylation. The molecular basis of this effect is not entirely understood. Binding of CaM to the phosphorylated and the unphosphorylated states of CaMKII is well mimicked by the interaction of CaM with two different length peptides taken from the CaM-binding region of CaMKII, peptides we refer to as the long and intermediate peptides. To better understand the conformational change accompanying CaM trapping, we have used isothermal titration calorimetry (ITC) to compare the binding thermodynamics of CaM to these peptides as well as to a shorter CaMKII-based peptide. Calorimetric analysis revealed that the enthalpy, rather than the entropy, distinguished binding of these three peptides. Furthermore, the heat capacity change was found to be similar for the long and intermediate peptides but smaller in magnitude for the short peptide. Direct titration of CaM with peptide provided the Kd value for the short peptide (Kd = 5.9 +/- 2.4 microM), but a novel, two-phased competitive binding strategy was necessary to ascertain the affinities of the intermediate (Kd = 0.17 +/- 0.06 nM) and long (Kd = 0.07 +/- 0.04 pM) peptides. To our knowledge, the Kd for the long peptide is the most potent measured to date using ITC. Together, the findings reported here support a model whereby the final conformational change accompanying CaM trapping buries little additional surface area but does involve formation of new hydrogen bonds and van der Waals contacts that contribute to formation of the high-affinity, CaM-trapped state.

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.

The transferrins (TF) are a family of bilobal glycoproteins that tightly bind ferric iron. Each of the homologous N- and C-lobes contains a single iron-binding site situated in a deep cleft. Human serum transferrin (hTF) serves as the iron transport protein in the blood; circulating transferrin binds to receptors on the cell surface, and the complex is internalized by endocytosis. Within the cell, a reduction in pH leads to iron release from hTF in a receptor-dependent process resulting in a large conformational change in each lobe. In the hTF N-lobe, two critical lysines facilitate this pH-dependent conformational change allowing entry of a chelator to capture the iron. In the C-lobe, the lysine pair is replaced by a triad of residues: Lys534, Arg632, and Asp634. Previous studies show that mutation of any of these triad residues to alanine results in significant retardation of iron release at both pH 7.4 and pH 5.6. In the present work, the role of the three residues is probed further by conversion to the residues observed at the equivalent positions in ovotransferrin (Q-K-L) and human lactoferrin (K-N-N) as well as a triad with an interchanged lysine and arginine (K534R/R632K). As expected, all of the constructs bind iron and associate with the receptor with nearly the same K(D) as the wild-type monoferric hTF control. However, interesting differences in the effect of the substitutions on the iron release rate in the presence and absence of the receptor at pH 5.6 are observed. Additionally, titration with KCl indicates that position 632 must have a positively charged residue to elicit a robust rate acceleration as a function of increasing salt. On the basis of these observations, a model for iron release from the hTF C-lobe is proposed. These studies provide insight into the importance of charge and geometry of the amino acids at these positions as a partial explanation for differences in behavior of individual TF family members, human serum transferrin, ovotransferrin, and lactoferrin. The studies collectively highlight important features common to both the N- and C-lobes of TF and the critical role of the receptor in iron release.

Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site > or =30 A from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.

Transferrin receptor (TfR) is a dimeric cell surface protein that binds both the serum iron transport protein transferrin (Fe-Tf) and HFE, the protein mutated in patients with the iron overload disorder hereditary hemochromatosis. HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, but HFE binding to TfR lowers the apparent affinity of the Fe-Tf/TfR interaction. This apparent affinity reduction could result from direct competition between HFE and Fe-Tf for their overlapping binding sites on each TfR polypeptide chain, from negative cooperativity, or from a combination of both. To explore the mechanism of the affinity reduction, we constructed a heterodimeric TfR that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Binding studies using a heterodimeric form of soluble TfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of the effects of HFE on iron homeostasis result from competition with Fe-Tf for TfR binding. Experiments using transfected cell lines demonstrate a physiological role for this competition in altering HFE trafficking patterns.

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf., Competing Interests: The authors have declared that no conflicts of interest exist.

Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.

The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR., (Copyright 2001 Academic Press.)

We have used X-ray fiber diffraction to probe the structure of fibers of tau and tau fragments. Fibers of fragments from the microtubule binding domain had a cross beta-structure that closely resembles that reported both for neurofibrillary tangles found in Alzheimer's disease brain and for fibrous lesions from other protein folding diseases. In contrast, fibers of full-length tau had a different, more complex structure. Despite major differences at the molecular level, all fiber types exhibited very similar morphology by electron microscopy. These results have a number of implications for understanding the etiology of Alzheimer's and other tauopathic diseases. The morphology of the peptide fibers suggests that the region in tau corresponding to the peptides plays a critical role in the nucleation of fiber assembly. The dramatically different structure of the full length tau fibers suggests that some region in tau has enough inherent structure to interfere with the formation of cross beta-fibers. Additionally, the similar appearance by electron microscopy of fibrils with varying molecular structure suggests that different molecular arrangements may exist in other samples of fibers formed from tau.