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.

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. Purified CETP itself bound weakly to LPS with a Kd ≥ 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-α normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE 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.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor 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.

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.

beta- 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 beta peptide populations were extracted from animal brain tissue or cell- conditioned media, immunoprecipitated with specific antibodies, and analyzed by matrixassisted laser desorption time- of- flight mass spectrometry. N-15- Substituted A beta 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 beta 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 beta peptide profile of an inhibitor of gamma-secretase, one of the two proteinases that excises A beta 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.

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 Kd = 170 nM at the neutral pH of plasma, but with a Kd 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 ∼5-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.