Prostatic acid phosphatase (PAP, also known as ACPP) has been used for over 50 years as a prostate cancer biomarker.1
Recently, we found that the transmembrane isoform of PAP was expressed in nociceptive (pain-sensing) dorsal root ganglia neurons.2
In addition, we found that the secretory and transmembrane isoforms of PAP have ectonucleotidase activity, as demonstrated by the ability of each isoform to dephosphorylate extracellular AMP to adenosine. In mice, intrathecal injection of the secretory isoform of PAP has antinociceptive effects in chronic inflammatory and neuropathic pain models that persist for three days.2
These antinociceptive effects were entirely dependent on adenosine A1
receptor activation, suggesting PAP generates adenosine in vivo
Intrathecal injections are routinely performed in humans but they are invasive. Moreover, it is impractical to deliver PAP protein orally because, like all proteins, PAP is susceptible to proteolysis in the digestive tract. In contrast, small molecule activators of PAP could enhance the activity of endogenously expressed PAP and be made orally bioavailable. At present, there are no known small molecule activators of PAP.
Conversely, it would be advantageous to have small molecules that selectively and acutely inhibit PAP for physiological studies. L-(+)-tartrate is the most commonly used inhibitor of PAP but must be used at high (mM) concentrations. Also, L-(+)-tartrate inhibits other acid phosphatases.3
α-Benzylaminobenzylphosphonic acid (BABPA), a derivative of benzylphosphonic acid, was found to inhibit PAP in the low nanomolar range.4
While potent, this inhibitor is not routinely used because it is difficult to obtain (not commercially available and must be synthesized). It is unknown if BABPA inhibits other phosphatases. Discovering selective PAP inhibitors would provide a way to examine the acute biological effects of PAP inhibition and could complement studies with Pap−/−
mice where enzyme activity is eliminated throughout life.2
We previously identified several structurally diverse inhibitors of hPAP using a high-throughput 384-well single point fluorogenic screen.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.6
We screened the Library of Pharmacologically Active Compounds (LOPAC1280
) as an eight-point dilution series and identified several potential hPAP inhibitors. Three of these compounds also inhibited hPAP and recombinant mPAP in an orthogonal biochemical assay. This orthogonal assay used AMP, a physiologically-relevant PAP substrate. Of these compounds, pCPT-cAMP and a related analog (pCPT-cGMP) were the only compounds that inhibited PAP in a real-time cell-based calcium mobilization assay. These cyclic nucleotide analogs are the first known compounds that inhibit PAP in vitro
as well as in living cells.