Brian J. Stockman

SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1

Sirtuins catalyze NAD+-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1–7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

WaterLOGSY as a method for primary NMR screening: Practical aspects and range of applicability

WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.

Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: The A-state of human ubiquitin

SummaryHuman ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitins role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly 15N-enriched ubiquitin. Sequence-specific 1H and 15N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets. Because of 1H chemical shift degeneracies, the increased resolution provided by the 15N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of β-sheet, comprising residues 2–17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an α-helix, comprising residues 25–33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of β-sheet. It is likely, therefore, that residues 1–33 comprise a folded domain in the A-state of ubiquitin. On the basis of 1Hα chemical shifts and weak short-range NOEs, residues 34–76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional 1H methods [Harding et al. (1991) Biochemistry, 30, 3120–3128].

Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures.

High-throughput ligand-based NMR screening with competition binding experiments is extended to (19)F detection. Fluorine is a favorable nucleus for these experiments because of the significant contribution of the Chemical Shift Anisotropy (CSA) to the (19)F transverse relaxation of the ligand signal when bound to a macromolecular target. A low to moderate affinity ligand containing a fluorine atom is used as a reference molecule for the detection and characterization of new ligands. Titration NMR experiments with the selected reference compound are performed for finding the optimal set-up conditions for HTS and for deriving the binding constants of the identified NMR hits. Rapid HTS of large chemical mixtures and plant or fungi extracts against the receptor of interest is possible due to the high sensitivity of the (19)F nucleus and the absence of overlap with the signals of the mixtures to be screened. Finally, a novel approach for HTS using a reference molecule in combination with a control molecule is presented.

Identification of Allosteric PIF-Pocket Ligands for PDK1 using NMR-Based Fragment Screening and 1H-15N TROSY Experiments

Aberrant activation of the phosphoinositide 3‐kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3‐phosphoinositide‐dependent kinase‐1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N‐terminal lobe of the 3‐phosphoinositide‐dependent kinase‐1 catalytic domain contains a docking site which recognizes the non‐catalytic C‐terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so‐called PDK1 Interacting Fragment pocket allows interaction with 3‐phosphoinositide‐dependent kinase‐1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3‐phosphoinositide‐dependent kinase‐1 domain construct against a library of chemically diverse fragments in order to identify small, ligand‐efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP‐site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand‐induced changes in 1H‐15N TROSY spectra acquired using uniformly 15N‐enriched PDK1 provided evidence to distinguish ATP‐site from PDK1 Interacting Fragment‐site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.

Competition Binding Experiments for Rapidly Ranking Lead Molecules for their Binding Affinity to Human Serum Albumin

Many lead molecules that have high affinity for a therapeutic target in vitro exhibit a reduced efficacy in vivo. Drug binding to human serum albumin is a major contributor to this reduction in potency, and many drug discovery programs expand significant resources preparing compounds that have decreased albumin binding. As rational and structure-based approaches have already been demonstrated to design compounds that have reduced affinity for albumin, the ability to rapidly and accurately assess protein binding will be valuable in lead optimization. This review will summarize some of the NMR-based efforts towards developing universal, rapid, accurate, and site-specific assays for estimating protein binding.

Dynamics of stromelysin/inhibitor interactions studied by 15N NMR relaxation measurements: Comparison of ligand binding to the S1-S3 and S′1-S′3 subsites

This report describes the backbone amide dynamics of the uniformly 15N labeled catalytic domain of human stromelysin complexed to PNU-99533, a hydroxamate-containing ligand that binds to the S′1-S′3 region (right side) of the stromelysin active site, and to PNU-107859 and PNU-142372, both thiadiazole-containing ligands that bind to the S1-S3 region (left side) of the stromelysin active site. 15N R1, R2 and NOE NMR relaxation measurements were recorded and analyzed for each complex. Different dynamic behaviors were observed for stromelysin complexed to the two classes of ligands, indicating that it may be possible to use protein dynamics to distinguish between different binding orientations. In the absence of bound ligand at the S1-S3 subsites, the S1-S3 residues were found to be relatively rigid. In contrast, the S′1-S′3 subsites were found to be flexible in the absence of interactions with ligand. The relative rigidness of the S1-S3 subsites may be responsible for MMP binding specificity by discriminating between ligands of different shapes. By contrast, the inherent flexibility of the S′1-S′3 subsites allows structural rearrangement to accommodate a broad range of incoming substrates or inhibitors. Similarities and differences in dynamics observed for each complex provide insights into the interactions responsible for protein–ligand recognition. The relevance of protein dynamics to structure-based drug design is discussed.

2-Fluoro-ATP as a versatile tool for 19F NMR-based activity screening.

19F NMR-based methods have found utility in activity-based screening assays. However, because enzymes catalyze a diverse set of reactions, a large variety of fluorinated substrates would need to be identified to target each one separately. We have developed a more streamlined approach that is applicable to many enzymes that utilize ATP as a substrate. In this method, a fluorine-containing ATP analogue, 2-fluoro-ATP, is used to monitor the reaction. Applications are described for nicotinamide adenine dinucleotide synthetase and 3-phosphoinositide dependent kinase-1. Fragment screening results for the latter indicate that this technique can identify compounds that inhibit as well as activate reactions. The present results, together with previous biochemical studies from other laboratories, have shown that 2-fluoro-ATP can serve as a substrate for nine enzymes that are representative of three of the six enzyme subclasses, namely the transferases, hydrolases, and ligases. This suggests that 2-fluoro-ATP is suitable as a universal tool for screening ATP-requiring enzymes. Importantly, 2-fluoro-ATP has been determined to be a valid substrate for a variety of kinases, including both small molecule and protein kinases, suggesting that it may be useful for investigating the large number of pharmaceutically relevant kinases.

Redox and spectral properties of flavodoxin from Anabaena 7120

We report here on the spectrophotometric and electrochemical properties of the flavodoxin from Anabaena 7120 and compare these properties with those of flavodoxins that have been studied previously. Molar absorption coefficients have been determined for all three oxidation states of this protein, at various wavelengths. For oxidized flavodoxin, molar absorption coefficients for the absorption maxima at 464 and 373 nm were 9200 and 8500 M-1 cm-1, respectively. Reduction by the first electron produced a neutral blue semiquinone which exhibited an absorption maximum at 575 nm. The molar absorption coefficients at 575 nm were 200 M-1 cm-1 for the oxidized form, 5100 M-1 cm-1 for the semiquinone form, and 250 M-1 cm-1 for the hydroquinone form. Redox potentials have been determined, in the pH range of 6.0 to 8.5, for both electron transfers. At pH 7.0, the midpoint potential values for the first and second electron transfers were -0.196 and -0.425 V, respectively. We determined that the first electron transfer is pH dependent and that a proton transfer accompanies this one electron transfer. It was also determined that the second electron transfer is pH independent in the pH range of 6.0 to 8.5.

Multi-Selective One Dimensional Proton NMR Experiments for Rapid Screening and Binding Affinity Measurements

High-throughput ligand-based proton NMR screening performed in the presence of a spy molecule and a control molecule is a valuable tool for identifying drug leads. A limitation of the technique is represented by the severe overlap encountered in the screening of large chemical mixtures. An approach for overcoming this overlap problem is the use of multi-selective R(1) filtered and COSY or TOCSY experiments. Application of this methodology to compounds binding to the Sudlow site I of human serum albumin is presented. The screening is performed by simply monitoring the intensity of two signals. The precise measurement of the relative intensity of the two resonances permits determination of the binding constant of the NMR-hit. For a simple competition binding mechanism, the rapidly-derived NMR binding constants are in good agreement with the values derived from full-titration ITC and fluorescence spectroscopy measurements.