A High Throughput Assay to Identify Small Molecule Modulators of Prostatic Acid Phosphatase

Abstract

Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. Injection of the secretory isoform of PAP has potent antinociceptive effects in mouse models of chronic pain. These data suggested that a small molecule activator of PAP may have utility as a novel therapeutic for chronic pain, while inhibitors could be used to acutely inhibit PAP in vitro and in vivo. To identify small molecule modulators of PAP activity, we validated a high throughput, fluorescence-based biochemical assay and then used this assay to screen a compound library. We decreased the frequency of false positive activators by subtracting compound fluorescence from the final assay fluorescence. This approach significantly reduced the number of false positive activators found in the screen. While no activators were confirmed, seven novel inhibitors of PAP were identified. Our results suggest this high throughput assay could be used to identify small molecule modulators of PAP activity.

Up-regulated Ectonucleotidases in Fas-Associated Death Domain Protein– and Receptor-Interacting Protein Kinase 1–Deficient Jurkat Leukemia Cells Counteract Extracellular ATP/AMP Accumulation via Pannexin-1 Channels during Chemotherapeutic Drug-Induced Apoptosis

“…Up-regulated PAP is also used as a biomarker of prostate cancer (Hakalahti et al, 1993), and our observations suggest the possibility that it may also be a marker for some subtypes of leukemias. Because selective and well-defined pharmacologic inhibitors of PAP ectonucleotidase activity are not readily available (Larsen et al, 2009;McCoy et al, 2013), most studies have used PAP-knockout mice to define physiologic roles (Zylka et al, 2008;Street et al, 2011;Yegutkin et al, 2014). Studies using siRNA with Jurkat cells and other human leukemia lines would be informative to define roles for TM-PAP in the growth, survival, and immunogenicity of hematopoietic tumors.…”

High-Throughput Screen Identifies Cyclic Nucleotide Analogs That Inhibit Prostatic Acid Phosphatase

“…5 Compound 49 inhibited AMP hydrolysis by hPAP and mPAP with an IC 50 of 2.90 μM for hPAP and 42.9 μM for mPAP (Fig 2E) but did not inhibit ALP (Fig 2F). As a second inhibitor control, we used L-(+)-tartrate (10 mM), which has a low affinity for PAP and must be used at high concentrations to inhibit PAP enzymatic activity.…”

“…We were similarly unable to identify hPAP activators in an end-point screen of 28,800 small molecules. 5 These results suggest that the odds of identifying hPAP activators are extremely low. In contrast, three of the candidate inhibitors (pCPT-cAMP, calmidazolium chloride, and nalidixic acid) identified in our present screen selectively inhibited hPAP and mPAP (but not pAP or ALP) in an orthogonal AMP hydrolysis assay.…”

“…5 Here, we modified this single-point assay so that we could perform a quantitative (multi-concentration) high-throughput screen in 1,536 well plates with secretory hPAP and DiFMUP as substrate. Quantitative HTS has several advantages over traditional single point screens, including fewer false negatives and generation of dose-response curves as part of the screen.…”

A High Throughput Assay for Discovery of Small Molecules that Bind AMP-activated Protein Kinase (AMPK)

“…One substantial drawback to using MANT-ADP as a fluorescent probe is the potential for false negatives due to the high percentage of autofluorescent compounds near 460 nm [ 29 - 31 ]. Thus, our assay cannot distinguish between fluorescent true negatives and fluorescent false negatives that inhibit binding of MANT-ADP to AMPK.…”