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.

Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with K(d) = 170 nM at the neutral pH of plasma, but with a K(d) as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.

We have identified a series of potent cholesteryl ester transfer protein (CETP) inhibitors, one member of which, torcetrapib, is undergoing phase 3 clinical trials. In this report, we demonstrate that these inhibitors bind specifically to CETP with 1:1 stoichiometry and block both neutral lipid and phospholipid (PL) transfer activities. CETP preincubated with inhibitor subsequently bound both cholesteryl ester and PL normally; however, binding of triglyceride (TG) appeared partially reduced. Inhibition by torcetrapib could be reversed by titration with both native and synthetic lipid substrates, especially TG-rich substrates, and occurred to an equal extent after long or short preincubations. The reversal of TG transfer inhibition using substrates containing TG as the only neutral lipid was noncompetitive, suggesting that the effect on TG binding was indirect. Analysis of the CETP distribution in plasma demonstrated increased binding to HDL in the presence of inhibitor. Furthermore, the degree to which plasma CETP shifted from a free to an HDL-bound state was tightly correlated to the percentage inhibition of CE transfer activity. The finding by surface plasmon resonance that torcetrapib increases the affinity of CETP for HDL by z5-fold likely represents a shift to a binding state that is nonpermissive for lipid transfer. In summary, these data are consistent with a mechanism whereby this series of inhibitors block all of the major lipid transfer functions of plasma CETP by inducing a nonproductive complex between the transfer protein and HDL. Despite the demonstration of the atheroprotective effects of HDL over the past several decades (1-4), no current therapy exists that is effective and well tolerated for increasing the levels of this lipoprotein (5). Although the use of extended-release niacin (Niaspan) at daily doses of 2 g or less has served to minimize the high incidence of vasodilatory effects, such as flushing and itching (6), toleration issues continue to limit compliance. Also, at 2 g/day, niacin increases high density lipoprotein cholesterol (HDL-C) by ,30% (7). The high levels of HDL associated with human cholesteryl ester transfer protein (CETP) deficiency (8) have suggested CETP inhibition as a means of increasing HDL. Although expression of human CETP in transgenic mice has produced mixed results regarding its atherogenicity, more consistent antiatherogenic effects have resulted from the inhibition of endogenous CETP in rabbits (8). In the wake of the beneficial effects observed through CETP inhibition in rabbits by induction of autoantibodies (9) and by administration of a synthetic inhibitor (10), these interventions have progressed to trials aimed at increasing HDL in humans. Although the use of the CETP vaccine has yet to demonstrate sufficient anti-CETP response to increase HDL (11), 900 mg/day of the inhibitor JTT-705 increased HDL-C by 34% and decreased low density lipoprotein cholesterol (LDL-C) by 7% (12).We have identified a new series of CETP inhibitors culminating in the dev...

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, nonreceptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.Proline-rich tyrosine kinase 2 (PYK2) 2 and focal adhesion kinase (FAK) comprise the focal adhesion kinase subfamily of non-receptor tyrosine kinases. PYK2 and FAK are large multidomain proteins containing an N-terminal FERM domain, a central catalytic domain, and a C-terminal segment containing dual proline rich (PR) subdomains and a focal adhesion targeting (FAT) region (1, 2). While FAK is widely expressed, PYK2 expression is relatively restricted with highest levels in brain and the hematopoeitic system. Unlike FAK, optimal PYK2 activation is dependent on Ca 2ϩ mobilization. PYK2 (Ϫ/Ϫ) animals have been described previously, and develop normally (3, 4). Characterization of the immune system of PYK2(Ϫ/Ϫ) animals revealed the absence of marginal zone B-cells along with abnormal T-cell independent type II responses (4), and altered macrophage morphology, migration and signaling in response to cell attachment or chemokine treatment (3). These studies strengthen the link between PYK2 and signaling through chemokine and integrin receptors. In addition, PYK2(Ϫ/Ϫ) mice were shown to have increased susceptibility to diet-induced obesity and diabetes (5).Recently, the characterization of PYK2(Ϫ/Ϫ) mice showed a high bone mass phenotype resulting from increased osteogenesis and osteoblast activity. Using PYK2(Ϫ/Ϫ) mouse bone marrow cultures and hMSCs expressing a PYK2 shRNA, elimination or reduction of PYK2 protein levels resulted in significantly enhanced osteogeogenesis. Importantly, the daily administration of a pyrimidine-based PYK2 inhibitor, PF-431396, increased bone formation, and protected against bone loss in ovariectomized rats (6). PYK2(Ϫ/Ϫ) mice showed mild osteopetrosis which was attributed to the impairment in osteoclast function (7). Therefore, the high bone mass phenotype may result from both enhanced osteoblast and impaired osteocla...

to identify agents for raising the levels of HDL. To date, the most potent means for raising plasma HDL has been through the use of cholesteryl ester transfer protein (CETP) inhibitors. Both torcetrapib ( 1, 2 ) and anacetrapib ( 3 ) have demonstrated 2-fold elevations in human trials. However, the development of these new lipid-modulating drugs suffered a major setback in December 2006 with the termination of the phase 3 trials of torcetrapib due to an excess in overall mortality and adverse cardiovascular events ( 4, 5 ). The largest of these trials, the Investigation of Lipid Level Management to Understand Its Impact in Atherosclerotic Events (ILLUMINATE), involved 15,067 patients at high cardiovascular risk. At study termination, 93 deaths had occurred in the torcetrapib/atorvastatin group compared with 59 in atorvastatin group. Of the 34 excess deaths in the torcetrapib group, 14 were cardiovascular-related whereas 20 were noncardiovascular-related. Although the occurence of new infections during the trial was similar for the torcetrapib versus the atorvastatin group (182 versus 177), there were nine deaths from infection in the former group versus zero for the latter.That nearly half of the noncardiovascular excess in mortality for the ILLUMINATE trial was associated with infection raises the question as to whether torcetrapib, apart from its intended effect on CETP, might have interfered with the function of two other proteins in the same family of lipid binding proteins, both of which play important roles in antibacterial defense ( 6 ). The fi rst of these, lipopolysaccharide binding protein (LBP), is an acute phase Abstract The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purifi ed CETP itself bound weakly to LPS with a Kd у 25 uM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-␣ normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMI-NATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis. In order to reduce the incidence of cardiovascular disease beyond that achieved through the use of sta...

Cholesteryl ester transfer protein (CETP) transfers neutral lipids between different types of plasma lipoprotein. Inhibitors of CETP elevate the fraction of plasma cholesterol associated with high-density lipoproteins and are being developed as new agents for the prevention and treatment of cardiovascular disease. The molecular basis of their function is not yet fully understood. To aid in the study of inhibitor interactions with CETP, a torcetrapib-related compound was coupled to different biotin-terminated spacer groups, and the binding of CETP to the streptavidin-bound conjugates was monitored on agarose beads and in a surface plasmon resonance biosensor. CETP binding was poor with a 2.0 nm spacer arm, but efficient with polyethyleneglycol spacers of 3.5 or 4.6 nm. The conjugate based on a 4.6 nm spacer was used for further biosensor experiments. Soluble inhibitor blocked the binding of CETP to the immobilized drug, as did preincubation with a disulfide-containing covalent inhibitor. To provide a first estimate of the binding site for torcetrapib-like inhibitors, CETP was modified with a disulfide-containing agent that modifies Cys-13 of CETP. Mass spectrometry of the modified protein indicated that a single half-molecule of the disulfide was covalently bound to CETP, and peptide mapping after digestion with pepsin confirmed previous reports based on mutagenesis that Cys-13 was the site of modification. Modified CETP was unable to bind to the biosensor-mounted torcetrapib analog, indicating that the binding site on CETP for torcetrapib is in the lipid-binding pocket near the N-terminus of the protein. The crystal structure of CETP shows that the sulfhydryl group of Cys-13 resides at the bottom of this pocket.

␤-Amyloid peptides, tentatively regarded as the principal neurotoxins responsible for Alzheimer's Disease, make up a set of products that varies significantly among different biological systems. The full implications of this complexity and its variations have yet to be defined. In this work, A␤ peptide populations were extracted from animal brain tissue or cell-conditioned media, immunoprecipitated with specific antibodies, and analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. 15N-Substituted A␤ internal standards were added to gauge variations in the profile of captured peptides. Results from a range of species, including guinea pig, dog, rabbit, and wild-type and transgenic mice, showed that the A␤ peptide population in each system was mainly determined by the species of origin of the amyloid precursor protein (APP) and not by the host tissue or cell line. The same method was used to gauge the effect on the A␤ peptide profile of an inhibitor of ␥-secretase, one of the two proteinases that excises A␤ peptides from the precursor protein with different effects on specific peptides. Overall, the results demonstrate that the species of origin of the APP substrate dictates the outcome of APP processing to a greater extent than the origin of the processing enzymes, an important consideration in rationalizing the properties of different model systems.A␤ peptides are tentatively regarded as the principal neurotoxins responsible for AD (Watson et al., 2005) and are formed by sequential proteolytic events near the C terminus of the 770-residue APP. They are also the main components of amyloid plaques that are the classical biomarker of AD in post mortem brain. In the first step of pathological processing, the membrane-bound aspartic proteinase BACE cleaves APP after Met-671 to generate a C-terminal fragment called APP-C-terminal fragment ␤. A␤ species are liberated from this fragment by the intramembrane proteinase ␥-secretase. Both secretases are seen as important drug targets, because the amyloid hypothesis of AD implies that blocking the production of A␤ peptides will retard the disease (Gandy, 2005). The alternative ␣-secretory pathway does not lead to A␤ peptide production.