A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation

Sirtuins are important regulators of lysine acylation, which is implicated in cellular metabolism and transcriptional control. This makes the sirtuin class of enzymes interesting targets for development of small molecule probes with pharmaceutical potential. To achieve detailed profiling and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2 as a long chain deacylase enzyme. Novel enzymatic activities of SIRT2 were thus established in vitro, which warrant further investigation, and two known inhibitors, suramin and SirReal2, were profiled against substrates containing ε-<i>N</i>-acyllysine modifications of varying length

Correction to A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation

Correction to A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluatio

Fluorescence polarization assay.

<p>A) assay principle; B) structures of the fluorescent AGP and HSA probes Dipyridamole and Dansyl sarcosine, respectively.</p

Structures of the AGP and HSA Validation Set.

<p>Structures of the AGP and HSA Validation Set.</p

Validation Set Testing.

<p>A) IC₅₀ and K_d values for the Validation Set in 1,536-well (▪), 96-well (▪), and protein coated bead (□) AGP assays; B) the corresponding results for the HSA assays.</p

HSA Assay Protocol for 1,536-well Format.

<p>HSA Assay Protocol for 1,536-well Format.</p

HSA and AGP IC₅₀ and K_d value comparisons for the 10-compound validation set.

<p>–: no activity exhibited.</p><p>NC: Ratio could not be calculated.</p>1<p>Based on literature.</p

Top LOPAC¹²⁸⁰ Hits.

1<p>Top compound concentrations of 144 or 151 µM for the AGP and HSA assays, respectively.</p

LOPAC¹²⁸⁰ Screen.

<p>The robust AGP (•) and HSA (o) screen performance as represented by the reproducible high Z’-factor trend.</p

Diminished Sensitivity of Chronic Lymphocytic Leukemia Cells to ABT-737 and ABT-263 Due to Albumin Binding in Blood

Purpose: Inhibition of the antiapoptotic BCL2 family is one of the most promising areas of anticancer drug development. However, ABT-737, a specific BCL2 inhibitor, is neither orally bioavailable nor metabolically stable. To overcome these problems, the structurally related molecule ABT-263 was synthesized and recently entered clinical trials in hematologic malignancies, including chronic lymphocytic leukemia (CLL). Almost all laboratory studies have been carried out with ABT-737 rather than ABT-263, the drug being used in clinical trials. Currently there are no published data on the comparative effects of these inhibitors. To gain insight into the potential value or limitations of ABT-263 in the clinic, we assessed its ability to induce apoptosis in clinically relevant cellular models of CLL. Experimental Design: The susceptibility of freshly isolated primary CLL cells to these inhibitors was compared in standard culture conditions and in conditions that more closely mimic in vivo conditions in a whole blood assay system. Results: ABT-737 was more potent than ABT-263 at inducing apoptosis in CLL cells. In whole blood, ∼100-fold higher concentrations of both drugs were required to induce apoptosis. We found that ABT-263 was highly bound by albumin and that an increased albumin binding of ABT-263 as compared with ABT-737 accounted for the differential sensitivity of CLL cells. Conclusions: Our data indicate that the exquisite in vitro sensitivity of CLL cells to BCL2 inhibitors may be lost in vivo due to high cell densities and the albumin binding of ABT-263. Modification of ABT-263 may yield a BCL2 inhibitor with greater bioavailability and more favorable pharmacokinetics