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WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3′ and 5′ ends. Ribonuclease P is the sole endonuclease responsible for processing the 5′ end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified 1 and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 Å resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA–RNA recognition by this ribozyme.

Ribonuclease (RNase) P is the universal ribozyme responsible for 5′-end tRNA processing. We report the crystal structure of the Thermotoga maritima RNase P holoenzyme in complex with tRNA Phe . The 154 kDa complex consists of a large catalytic RNA (P RNA), a small protein cofactor and a mature tRNA. The structure shows that RNA–RNA recognition occurs through shape complementarity, specific intermolecular contacts and base-pairing interactions. Soaks with a pre-tRNA 5′ leader sequence with and without metal help to identify the 5′ substrate path and potential catalytic metal ions. The protein binds on top of a universally conserved structural module in P RNA and interacts with the leader, but not with the mature tRNA. The active site is composed of phosphate backbone moieties, a universally conserved uridine nucleobase, and at least two catalytically important metal ions. The active site structure and conserved RNase P–tRNA contacts suggest a universal mechanism of catalysis by RNase P. Ribozyme RNaseP dissected Transfer RNAs are synthesized as precursors that require trimming at the 5' and 3' ends, and some modification of specific nucleotides. The ribozyme RNase P is universally responsible for processing the 5' end of tRNAs. The crystal structure of RNase P (from Thermotoga maritima ) bound to mature phenylalanine transfer RNA has now been solved. It reveals the interactions involved in pre-tRNA recognition, active site location and the role of metals in catalysis. The RNase P–tRNA ribonucleoprotein structure also offers clues as to how an ancient RNA-based world might evolve to become the protein-catalyst dominated world of today. tRNAs are synthesized in a premature form that requires trimming of the 5′ and 3′ ends and modification of specific nucleotides. RNase P, a complex containing a long catalytic RNA and a protein cofactor, catalyses the cleavage that generates the mature 5′ end. Here, the structure of RNase P bound to mature tRNA Phe is solved. Recognition of the leader sequence and its mechanism of cleavage is determined by soaking an oligonucleotide corresponding to the premature 5′ end into the crystal.

HU and IHF are members of a family of prokaryotic proteins that interact with the DNA minor groove in a sequence‐specific (IHF) or non‐specific (HU) manner to induce and/or stabilize DNA bending. HU plays architectural roles in replication initiation, transcription regulation and site‐specific recombination, and is associated with bacterial nucleoids. Cocrystal structures of Anabaena HU bound to DNA (1P71, 1P78, 1P51) reveal that while underlying proline intercalation and asymmetric charge neutralization mechanisms of DNA bending are similar for IHF and HU, HU stabilizes different DNA bend angles (∼105–140°). The two bend angles within a single HU complex are not coplanar, and the resulting dihedral angle is consistent with negative supercoiling. Comparison of HU–DNA and IHF–DNA structures suggests that sharper bending is correlated with longer DNA binding sites and smaller dihedral angles. An HU‐induced bend may be better modeled as a hinge, not a rigid bend. The ability to induce or stabilize varying bend angles is consistent with HU's role as an architectural cofactor in many different systems that may require differing geometries.

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor α-chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions including asthma. STAT6 binds poly(ADP-ribose) polymerase (PARP)14, an ADP-ribosyl monotransferase. Elimination of PARP14 by gene targeting led to attenuation of OVA-specific allergic lung inflammation. However, PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation, and it is not clear whether inhibition of the catalytic function has any biological consequence. Using BALB/c mice sensitized to the allergen Alternaria alternata, we show that peroral administration of RBN012759, a highly selective inhibitor of ADP-ribosylation by PARP14 with negligible impact on other members of the PARP gene family, achieved biologically active plasma concentrations and altered several responses to the Ag. Specifically, the pharmaceutical compound decreased mucus after allergen challenge, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that PARP14 catalytic activity can contribute to pathogenesis in allergic or atopic processes and propose that other biological endpoints dependent on ADP-ribosylation by PARP14 can be targeted using selective inhibition.

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor alpha chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions that include asthma. Prior work has shown a physical and functional association of STAT6 with PARP14, an ADP-ribosyl monotransferase. Moreover, elimination of all PARP14 expression by gene targeting led to altered recall antibody responses and attenuation of ovalbumin-specific allergic lung inflammation with no apparent health issues for mice lacking this protein. However, an unanswered question is whether or not inhibition of the catalytic function has any biological consequence since PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation. As reported separately, iterative structural analyses and medicinal chemistry fostered the generation of a compound, RBN2759, that is highly selective in its inhibition of PARP14 with negligible impact on other members of the PARP gene family. We show here that administration of this compound to mice previously sensitized to the allergen Alternaria alternata achieved biochemically active levels and altered physiological responses to the antigen. These results show for the first time that in vivo administration of a specific inhibitor of the ADP-ribosyltransferase activity encoded by PARP14 is sufficient to alter biological responses. Specifically, the orally absorbable pharmaceutical compound decreased allergen-induced mucus, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that the catalytic activity can contribute to pathogenesis in allergic processes and propose that other biological endpoints that depend on ADP-ribosylation by PARP14 can be targeted using selective inhibition. Competing Interest Statement All authors who are or were employees of Ribon Therapeutics, Inc. would benefit financially if the pharmaceutical agent that is central to this report, or derivatives thereof, were to make it to be an approved therapeutic. Thus, K. K., L. S., K. S., J. M., M. N., and H. K. are full-time employees of and hold equity interests in Ribon Therapeutics but were recused from analyses and interpretation of the data and from manuscript preparation; A. M. E., K. C., K. G., A. L. R., R. S. P., and S. H. C. report no potential conflicts of interest; M. R. B. holds equity in Regeneron, Inc., which markets a biologic agent used in treatment of allergic diseases and asthma.