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 fluorophorecontaining 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.

The results of a study to measure the beta-sheet forming propensities of the 20 naturally occurring amino acids are presented. The protein host for these studies is the 56 amino acid B1 domain of staphylococcal IgG binding protein G [Fahnestock, S.R., Alexander, P., Nagle, J., & Filpula, D. (1986) J. Bacteriol. 167, 870-880]. This protein was selected because it exhibits a reversible two-state thermal denaturation transition and its structure is known at high resolution. A suitable guest position in the protein was identified, and its neighboring environment was modified to minimize the potential for artifactual interactions. All 20 amino acids were individually substituted at the guest site, and their effect on the protein's thermal stability was determined. NMR was used to verify the structural integrity of several of the proteins with different amino acid substitutions at the guest site. The results of these studies provide a thermodynamic scale for the relative beta-sheet forming propensities of the amino acids that shows a clear correlation with the beta-sheet preferences derived from statistical surveys of proteins of known structure.

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

AMP-activated protein kinase (AMPK) is a principal metabolic regulator affecting growth and response to cellular stress. Comprised of catalytic and regulatory subunits, each present in multiple forms, AMPK is best described as a family of related enzymes. In recent years, AMPK has emerged as a desirable target for modulation of numerous diseases, yet clinical therapies remain elusive. Challenges result, in part, from an incomplete understanding of the structure and function of full-length heterotrimeric complexes. In this work, we provide the full-length structure of the widely expressed α1β1γ1 isoform of mammalian AMPK, along with detailed kinetic and biophysical characterization. We characterize binding of the broadly studied synthetic activator A769662 and its analogs. Our studies follow on the heels of the recent disclosure of the α2β1γ1 structure and provide insight into the distinct molecular mechanisms of AMPK regulation by AMP and A769662.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

Using site-directed mutagenesis in coijunction with NMR structural data on the adhesion domain of human CD2, we have defmed the binding region for CD58. Previous structural studies of rat and human CD2 indicate that this adhesion domain is immunoglobulin-like. Here we report that the CD58 binding site is a well-circumscribed, charged surface area covering -770 A2 on the AGFCC'C" face of the CD2 (8 barrel. This site contains 13-strand residues in the carboxyl-terminal half of the F strand (including Lys-82 and Tyr-86), the top of the C strand , and the C' strand (Gln-46), which are all solvent exposed. In addition, several exposed residues on the FG loop , the CC' loop (Lys-41 and Lys-43), and the C'C" loop (Arg-48 and Lys-51) form this site. In contrast, neither residues on the more peripheral G and C" strands of the same CD2 surface nor residues on B, E, and D strands of the opposite face are involved in CD58 binding. This CD58 binding site is predicted to lie most distal to the T-lymphocyte surface membrane, with ready access to CD58 on the surface of the opposing antigen-presenting cell.The interactions between helper T lymphocytes and their cognate partners (including antigen-presenting cells, epithelial cells, and endothelial cells) and between cytolytic T lymphocytes and target cells are predominantly mediated by adhesion molecules (reviewed in refs. 1 and 2). The transmembrane T-cell glycoprotein CD2 is an important component of these processes, functioning to promote the initial stages of cell contact even prior to recognition of antigen and major histocompatibility complex (MHC) by the T-cell receptor (TCR) (3, 4). CD2 is expressed on all T lymphocytes and the vast majority of thymocytes (5). Human CD2 binds to the cell surface glycoprotein CD58 (LFA-3), which is ubiquitously expressed on many cell types including antigenpresenting cells, thereby facilitating cell-cell adhesion (6, 7). The extracellular segment of CD2 comprises two domains with the membrane distal N-terminal domain mediating its adhesion function (8,9). Although the monomeric affinity of CD2 for CD58 is only micromolar, rapid CD58-dependent reorganization of CD2 at the interface between the T cell and the antigen-presenting cell readily establishes intercellular interactions via multipoint attachment (4, 10). Not surprisingly, antibodies directed against either CD2 or CD58 can block human T-cell activation (7).Recently, a detailed structure of the glycosylated adhesion domain ofhuman CD2 has been obtained by NMR and shows a typical immunoglobulin fold (11,12). This study complements and extends earlier studies on the structure of rat CD2The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (13,14). However, the human CD2 molecule differs from its rodent homologue in several major respects. First, the physiologic ligand of human CD2 is CD58, whereas the rat CD2 ligand is CD48 ...

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.