Use of Inosine Monophosphate Dehydrogenase Activity Assay to Determine the Specificity of PARP-1 Inhibitors

Abstract

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in purine nucleotide biosynthesis. It is responsible for catalyzing the oxidation of inosine monophosphate (IMP) into xanthosine monophosphate (XMP). Concurrently, the cofactor NAD is reduced to NADH. Poly(ADP-ribose) polymerase 1 (PARP-1) also utilizes NAD as a substrate to synthesize poly(ADP-ribose). It has been demonstrated that inhibition of PARP-1 activity can be an effective cancer therapeutic. However, most PARP-1 inhibitors, including olaparib, were developed as NAD analogs. Therefore, these inhibitors likely interfere with other NAD-dependent pathways such as the one involved in de novo purine metabolism. In this chapter, we describe a method to quantitatively measure IMPDH activity by taking advantage of the autofluorescence of the product NADH. We use this method to analyze the effects of olaparib and non-NAD-like PARP-1 inhibitor (5F02) on IMPDH activity. We found that olaparib, unlike 5F02, significantly inhibits IMPDH activity in a dose-dependent manner. Our results suggest that IMPDH inhibition is an off-target effect of olaparib treatment. The consequences of this effect should be addressed by future clinical studies.

Poly-ADP Ribosyl Polymerase 1 (PARP1) Regulates Influenza A Virus Polymerase

“…NP accumulation was reduced similar to knockdown of RNA binding proteins NPM1 and DDX17 that are known to be required for influenza virus polymerase activity [24]. The ribonucleotide synthesis enzyme inosine monophosphate dehydrogenase 2 (IMPDH2), a cellular factor that interacts with the PARP1 and PARP2 DDR proteome network [45, 49], was also required for influenza NP synthesis. Further study of PARP1 role in kinetics of viral RNA syntheses and expression of other viral proteins will require generation of viable knockout (CRISPR/Cas9) cell lines in PARP1 [23].…”

“…A similar increase in polymerase activity for PARG overexpression was observed at a high multiplicity of infection (MOI = 1 and MOI = 5), but this was not significant ( p <0.1), likely reflecting the incomplete efficiency of cDNA transfection. However, IMPDH2 that interacts with and supplies PARP1 and PARP2 with NAD+ substrates for enzymatic reactions [49] significantly enhances polymerase activity during infection (Figure 4(a)). These results illustrate the complexity of the IAV interaction with cellular PARylation pathways on the infected cell.…”