NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CβII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.

This article is available online at http://www.jlr.org between HDL and apoB-containing lipoproteins such as LDL, and is currently a target for increasing HDL cholesterol (HDL-C) and reducing LDL cholesterol (LDL-C). Small molecule inhibitors have been developed to inhibit CETP, including torcetrapib (Pfi zer), dalcetrapib (Roche), evacetrapib (Eli Lilly), and anacetrapib (ANA) (Merck). While initial clinical trials with torcetrapib established the validity of CETP inhibition as a statin-additive mechanism for reduction of LDL-C and elevation of HDL-C ( 1, 2 ), the phase III outcome trial ILLUMINATE demonstrated that torcetrapib treatment was associated with an increase in cardiovascular events, and overall mortality ( 3 ). A series of preclinical studies indicated that torcetrapib had compound-specifi c off-target activity that was unrelated to CETP inhibition ( 4-6 ). Dalcetrapib was evaluated in a large phase III clinical program (dal-HEART), however the phase III outcomes study (dal-OUTCOMES) was stopped early due to futility/lack of effi cacy ( 7,8 ). While a possible reason for a lack of effect on outcomes benefi t with dalcetrapib might be related to its weaker inhibition of CETP (manifest as an insuffi cient elevation of HDL-C or lack of an effect on LDL-C), the precise answer for why dalcetrapib was ineffective remains unknown.ANA is a potent CETP inhibitor which has not demonstrated the off-target activities of torcetrapib in both preclinical and clinical studies ( 9-11 ). In a recent 1.5 year safety study in ‫ف‬ 1,600 patients with cardiovascular disease ( 11 ), ANA treatment had no effect on blood pressure, electrolytes, or aldosterone, and the distribution of cardiovascular events suggested that ANA treatment would not be associated with the type of adverse effects on outcomes that were observed with torcetrapib. ANA treatment increases HDL-C by over 100% and lowers LDL-C by 30-40% as monotherapy and when coadministered with statins ( 9-11 ). In combination with the lack of any off-target, torcetrapib-like effects, Abstract Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased pre ␤ HDL-dependent cholesterol effl ux via ABCA1 in vitro. However, the effects of ANA on in vivo pre ␤ HDL have not been characterized. In vitro, ANA inhibited the formation of pre ␤ , however in ANA-treated dyslipidemic hamsters, pre ␤ HDL levels (measured by twodimensional gel electrophoresis) were increased, in contrast to in vitro fi ndings. Because changes in plasma pre ␤ HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamster...

Cholesteryl ester transfer protein (CETP) is a target for the treatment of dyslipidemia and coronary artery disease. In addition to the well-known effect of CETP to transfer CE from HDL to LDL and to VLDL, in vitro , CETP has been reported to transfer CE between small and large HDL particles (HDL2 and HDL3, respectively). We sought to understand how the CETP inhibitor anacetrapib (ANA) affects HDL3-to-HDL2 transfer under both in vitro and in vivo conditions. In vitro , ANA dose-dependently inhibited transfer of 3 H-CE from total HDL to LDL (IC50 30nM), and from isolated HDL3 to HDL2 particles (IC50 200nM). In human CETP transgenic mice, animals treated with a single dose of ANA (100mg/kg) displayed 80% maximal reduction in plasma CETP activity and a 22% increase in total HDL cholesterol. In animals treated with either vehicle or ANA, 3 H-CE-labeled HDL3 was injected intravenously and 3 H-tracer was monitored in lipoprotein fractions following injection. Animals treated with ANA showed an increase in the amount 3 H-tracer present in HDL2 compared to vehicle over time (20-70% increase across 6 hrs post 3 H-CE-HDL3 injection, P<0.05 vs vehicle). The HDL2 CE pool was also increased with ANA treatment, and 3 H-cholesterol flux into HDL2 was increased with ANA treatment when adjusted to the change in pool size (at 2 and 4 hrs post 3H-CE-HDL3 injection). No change in HDL2 3 H-tracer was seen in C57BL6 mice (lacking CETP) treated with ANA. These results indicate that in contrast to in vitro findings, ANA increases flux of CE into HDL2 in vivo , a process which likely involves multiple pathways. Therefore, the in vitro phenomena of 1) HDL3-to-HDL2 transfer by CETP and 2) inhibition of this transfer by CETP inhibitors are not recapitulated in vivo . It is clear that in vivo approaches are necessary to understand the relevance of HDL3-to-HDL2 transfer in vivo , and to accurately study the effects of CETP inhibition on lipoprotein metabolism.