Abstract
Poly(A)-specific ribonuclease (PARN) is an exoribonuclease/deadenylase that degrades 39-end poly(A) tails in almost all eukaryotic organisms. Much of the biochemical and structural information on PARN comes from the human enzyme. However, the existence of PARN all along the eukaryotic evolutionary ladder requires further and thorough investigation. Although the complete structure of the full-length human PARN, as well as several aspects of the catalytic mechanism still remain elusive, many previous studies indicate that PARN can be used as potent and promising anti-cancer target. In the present study, we attempt to complement the existing structural information on PARN with in-depth bioinformatics analyses, in order to get a hologram of the molecular evolution of PARNs active site. In an effort to draw an outline, which allows specific drug design targeting PARN, an unequivocally specific platform was designed for the development of selective modulators focusing on the unique structural and catalytic features of the enzyme. Extensive phylogenetic analysis based on all the publicly available genomes indicated a broad distribution for PARN across eukaryotic species and revealed structurally important amino acids which could be assigned as potentially strong contributors to the regulation of the catalytic mechanism of PARN. Based on the above, we propose a comprehensive in silico model for the PARN’s catalytic mechanism and moreover, we developed a 3D pharmacophore model, which was subsequently used for the introduction of DNP-poly(A) amphipathic substrate analog as a potential inhibitor of PARN. Indeed, biochemical analysis revealed that DNPpoly(A) inhibits PARN competitively. Our approach provides an efficient integrated platform for the rational design of pharmacophore models as well as novel modulators of PARN with therapeutic potential.
Citation: Vlachakis D, Pavlopoulou A, Tsiliki G, Komiotis D, Stathopoulos C, et al. (2012) An Integrated In Silico Approach to Design Specific Inhibitors Targeting Human Poly(A)-Specific Ribonuclease. PLoS ONE 7(12): e51113. doi:10.1371/journal.pone.0051113 Editor: Bruce R. Donald, Duke University Medical Center, Duke University, United States of America Received July 2, 2012; Accepted October 29, 2012; Published December 6, 2012 Copyright: ?2012 Vlachakis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist.
Introduction
The first and often rate-limiting step in eukaryotic mRNA turnover is the shortening of the poly(A) tail [1?]. The process is known as deadenylation and it occurs both in the nucleus and in the cytoplasm. In the nucleus it restricts newly added poly(A) tails to their appropriate lengths. In the cytoplasm, deadenylation either decreases the total mRNA levels and regulates the expression levels of specific mRNAs, or modulates the length of the poly(A) tail. Deadenylation is catalyzed by a family of specific ribonucleases, known as deadenylases [4?]. Among these, poly(A)-specific ribonuclease (PARN) has been involved in key biological processes, such as development, cell cycle progression, DNA damage response and cancer. PARN is conserved in many eukaryotes from yeast and plants to humans. PARN homologs are found in Schizosaccharomyces pombe (fission yeast) and Anopheles gambiae (mosquito), but they are notably absent from Saccharomyces cerevisiae and Drosophila melanogaster [5?], suggesting that they are not required by all eukaryotes [5]. Structural and biochemical studies revealed that PARN is homodimeric and the active site consists of four acidic amino acids Asp28, Glu30, Asp292, andAsp382, which are believed to coordinate the catalytically important divalent metal ions [8?]. Furthermore, the residue His377, which is conserved in PARN, has also been proposed to be essential for catalytic activity, thus classifying PARN as a DEDDh nuclease [9], named after the five conserved catalytic amino acid residues. The structure of PARN is composed of at least three functional domains: the catalytic nuclease domain, and two RNA binding domains: the R3H domain and the RNA binding domain or RNA recognition motif (RRM) [9?0] which have been suggested to contribute to the catalytic activity of the enzyme [9?2]. The RRM is a unique, multifunctional domain that is responsible for molecular recognition of the 59 cap structure [13]. The latter is perhaps the most characteristic feature of PARN that distinguishes it from all the other known deadenylases. Capbinding has been reported to significantly contribute to the processivity of the enzyme. Apart 59-cap, PARN activity is regulated by natural nucleotides [14?6] and by several protein factors. The latter include the cytoplasmic poly(A)-binding protein (PABPC) [17], the eukaryotic initiation factor 4E (eIF4E) [18] and the nuclear cap-binding complex (CBC) that negatively regulate PARN [19], while RHAU helicase [20] and AU-rich element(ARE) – binding proteins, including TTP and KSRP are positive regulators [21?3]. PARN activity is also regulated by factors that bind cytoplasmic polyadenylation elements (CPEs) including CPEbinding protein (CPEB) and the atypical Gld2 poly(A) polymerase [24,25]. Finally, PARN has been shown to be a target of synthetic nucleoside analogs with anticancer and antiviral potential. These analogs inhibit PARN activity in a competitive mode [26,27]. Furthemore, PARN mRNA and protein expression levels are elevated in acute leukemias [28]. These observations suggest that that enzyme may be a promising biomarker and a target for drug design [28]. Herein, we present a PARN-specific 3D pharmacophore model both for de novo design and virtual screening of selective inhibitors. For the design of the pharmacophore model, we initially used an in-depth phylogenetic analysis of PARN across species, which identified structurally conserved residues, important for the catalytic activity of the enzyme. Using a series of computer-aided molecular simulations, supported by statistical structure-activity correlations of our previously reported nucleoside analogs that inhibit PARN, we established a combined complex-based 3D pharmacophore model. We applied our in silico model to predict the effect of the amphipathic DNP-poly(A) substrate as a novel PARN-interacting molecule, which was then confirmed to efficiently inhibit the enzyme by kinetic assays.