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Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of precursor-tRNAs (pre-tRNA) and is conserved in all domains of life. However, the composition of RNase P varies from bacteria to archaea and eukarya, making RNase P one of the most diverse enzymes characterized. Most known RNase P enzymes contain a large catalytic RNA subunit that associates with one to 10 proteins. Recently, a protein-only form of RNase P was discovered in mitochondria and chloroplasts of many higher eukaryotes. This proteinaceous RNase P (PRORP) represents a new class of metallonucleases. Here we discuss our recent crystal structure of PRORP1 from Arabidopsis thaliana and speculate on the reasons for the replacement of catalytic RNA by a protein catalyst. We conclude, based on an analysis of the catalytic efficiencies of ribonucleoprotein (RNP) and PRORP enzymes, that the need for greater catalytic efficiency is most likely not the driving force behind the replacement of the RNA with a protein catalyst. The emergence of a protein-based RNase P more likely reflects the increasing complexity of the biological system, including difficulties in importation into organelles and vulnerability of organellar RNAs to cleavage.

Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana . PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.

Introduction: In the tumor micro-environment, extracellular ATP is sequentially hydrolyzed to adenosine by the ecto-nucleotidases CD39 (ATP→AMP) and CD73 (AMP→adenosine). Adenosine, through activation of the A2a receptor (A2aR), is a potent inhibitor of T-cell activation, resulting in an immunosuppressed phenotype. Thus, inhibition of A2aR has recently generated great interest in immuno-oncology. We present the characterization of a novel, selective, and highly potent small molecule antagonist of A2aR which is slated to enter the clinic in 2017. Methods: The cellular potency of A2aR antagonists was assessed as a function of decreased cAMP levels in CHO cells stably over-expressing hA2aR, a Gs coupled receptor, following stimulation with the agonist NECA. Experiments were conducted in the presence and absence of human serum. Selectivity against the Gi-coupled receptor A1R was assessed similarly as a function of cAMP elevation in CHO cells stably expressing hA1R, following pretreatment with forskolin and stimulation with NECA. The ability of AB928 to reverse adenosine-mediated immune suppression (25 μM) of human or mouse CD8+ T-cells was determined using standard CD3/CD28 activation conditions. CD25 expression and cytokine release were measured by flow cytometry and ELISA, respectively. The pharmacokinetic characteristics of AB928 were assessed in rodent and non-rodent species to facilitate calculation of a projected human dose. Results: AB928 represents a novel series of potent and selective compounds designed to inhibit adenosine-mediated A2aR activation. This molecule is different from most known A2aR antagonists in that it does not cross the blood brain barrier. AB928 inhibited NECA-mediated A2aR activation with a potency of Conclusions: AB928 is a potent, selective and peripherally restricted antagonist of the A2aR receptor which is slated to enter clinical development in 2017. Citation Format: Matt J. Walters, Joanne B. Tan, Annette Becker, Fangfang Yi, Tim Park, Manmohan R. Leleti, Brandon Rosen, Ehesan Sharif, Laurent Debien, Steve Young, Wan Hsin Lim, Stefan Garrido-Shaqfeh, Juan C. Jaen, Jay P. Powers. Characterization of the potent and selective A2aR antagonist AB928 for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2017-4572