High-Throughput Identification of Inhibitors of Human Mitochondrial Peptide Deformylase

Abstract

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.

Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

“…HsPDF elution was assessed by measuring the absorbance at 280 nm combined with measurements of HsPDF activity. HsPDF activity was assessed using a fluorescamine-based assay developed in our laboratory 45. Fractions containing HsPDF activity were pooled and digested with eight units of thrombin/mg of protein (Amersham Biosciences, Piscataway NJ) overnight at 16 °C.…”

Synthesis of Antiproliferative Cephalotaxus Esters and Their Evaluation against Several Human Hematopoietic and Solid Tumor Cell Lines: Uncovering Differential Susceptibilities to Multidrug Resistance

“…[17] As a result, deoxyharringtonine (2), anhydroharringtonine (5), and the b-lactone intermediate 79 (generated in the synthesis of 2, Scheme 13) were evaluated against a variety of human hematopoietic and solid tumor cell lines (Table 2). [96,97] These include HL-60 (acute promyelocytic leukemia), HL-60/RV + (a P-glycoprotein over-expressing multidrug resistant HL-60 variant which was selected by continuous exposure to the vinca alkaloid vincristine), JURKAT (T cell leukemia), ALL3 (acute lymphoblastic leukemia recently isolated from a patient treated at MSKCC and characterized as Philadelphia chromosome positive), NCEB1 (Mantle cell lymphoma), JEKO (B cell lymphoma), MOLT-3 (acute lymphoblastic T-cell), SKNLP (neuroblastoma), Y79 (retinoblastoma), PC9 (adenocarcinoma), H1650 (adenocarcinoma), H1975 (adenocarcinoma), H2030 (adenocarcinoma), H3255 (adenocarcinoma), TC71 (Ewings sarcoma), HTB-15 (glioblastoma), A431 (epithelial carcinoma), HeLa (cervical adenocarcinoma), and WD0082 (well-differentiated liposarcoma).…”

A Miniaturized 1536-Well Format γ-Secretase Assay

“…This similar hit rate was likely because these two validation libraries contain several pan-active compounds that have the potential to act as promiscuous active agents in the assay. 29,30 The Z′ values per formats exhibit similar signal to background ratios. Based on these optimization experiments, we set our fi nal assay conditions at 0.3 nM G2-10, 1 nM anti-mouse antibody, 15 nM streptavidin-XL665, and proceeded with the assay at 10 μL fi nal volume (5 μL γ-secretase mix + 5 μL HTRF mix) in the 1536-well format for screening.…”