High-throughput screen identifies cyclic nucleotide analogs that inhibit prostatic acid phosphatase

Area of Science:

• Biochemistry
• Pharmacology
• Molecular Biology

Background:

• Prostatic acid phosphatase (PAP) functions as an ectonucleotidase, dephosphorylating extracellular adenosine 5'-monophosphate (AMP) to adenosine.
• Currently, no known compounds effectively inhibit PAP activity within living cells, limiting therapeutic applications.

Purpose of the Study:

• To identify small-molecule inhibitors of both secretory and transmembrane prostatic acid phosphatase (PAP).
• To discover the first compounds capable of modulating PAP activity in live-cell assays.

Main Methods:

• A high-throughput screening (HTS) assay utilizing a fluorogenic substrate (difluoro-4-methylumbelliferyl phosphate) was employed to screen the Library of Pharmacologically Active Compounds (LOPAC(1280)).
• Candidate inhibitors were validated using an orthogonal absorbance-based biochemical assay with AMP as the substrate.
• Cell-based assays were conducted to assess the efficacy of identified compounds against transmembrane PAP activity.

Main Results:

• Three compounds, 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid, were identified as inhibitors of secretory human and mouse PAP.
• These compounds did not exhibit inhibitory activity against recombinant alkaline phosphatase.
• Only pCPT-cAMP and its analog 8-[4-chlorophenylthio] cGMP (pCPT-cGMP) demonstrated inhibition of transmembrane PAP ectonucleotidase activity in cell-based assays.

Conclusions:

• Two cyclic nucleotide analogs, pCPT-cAMP and pCPT-cGMP, were identified as effective inhibitors of both secretory and transmembrane PAP.
• These compounds represent the first small molecules found to inhibit PAP in vitro and in live cells.
• The identified inhibitors offer potential for developing novel therapeutic strategies targeting PAP-related conditions.