Cyclic peptides target the aromatic cage of a PHD-finger reader domain to modulate epigenetic protein function.

Plant homeodomain fingers (PHD-fingers) are a family of reader domains that can recruit epigenetic proteins to specific histone modification sites. Many PHD-fingers recognise methylated lysines on histone tails and play crucial roles in transcriptional regulation, with their dysregulation linked to various human diseases. Despite their biological importance, chemical inhibitors for targeting PHD-fingers are very limited. Here we report a potent and selective de novo cyclic peptide inhibitor (OC9) targeting the Nε-trimethyllysine-binding PHD-fingers of the KDM7 histone demethylases, developed using mRNA display. OC9 disrupts PHD-finger interaction with histone H3K4me3 by engaging the Nε-methyllysine-binding aromatic cage through a valine, revealing a new non-lysine recognition motif for the PHD-fingers that does not require cation-π interaction. PHD-finger inhibition by OC9 impacted JmjC-domain mediated demethylase activity at H3K9me2, leading to inhibition of KDM7B (PHF8) but stimulation of KDM7A (KIAA1718), representing a new approach for selective allosteric modulation of demethylase activity. Chemoproteomic analysis showed selective engagement of OC9 with KDM7s in T cell lymphoblastic lymphoma SUP T1 cells. Our results highlight the utility of mRNA-display derived cyclic peptides for targeting challenging epigenetic reader proteins to probe their biology, and the broader potential of this approach for targeting protein-protein interactions.

The influence of degree of labelling upon cellular internalisation of antibody-cell penetrating peptide conjugates.

Antibody-based agents are increasingly used as therapeutics and imaging agents, yet are generally restricted to cell surface targets due to inefficient cellular internalisation and endosomal entrapment. Enhanced cell membrane translocation of antibodies can be achieved by the covalent attachment of cell-penetrating peptides, including the HIV-1-derived transactivator of transcription (TAT) peptide. This study evaluated the cellular internalisation properties of five anti-HER2 Herceptin-TAT conjugates with degrees of TAT labelling (DOLTAT) ranging from one to five. Herceptin-TAT conjugates were synthesised via a strain-promoted alkyne-azide cycloaddition reaction, characterised by UV-vis spectroscopy, MALDI-TOF, and gel electrophoresis, then radiolabelled with zirconium-89 to permit measurement of cellular internalisation by gamma counting. [89Zr]Zr-DFO-Her-TAT(0-5) conjugates were isolated in high radiochemical purity (>99%) and exhibited high stability in murine and human serum over 7 days at 37 °C. Significant increases in cellular internalisation were observed for [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT values of 2 and above in SKBR3 (high HER2) cells over 48 h, in contrast to low-level non-specific uptake in MDA-MB-468 (low HER2) cells that did not increase over time. Notably, [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT of 3, 4, and 5 reached uptake values in SKBR3 cells of 5, 6, and 8% of the applied dose at 48 h respectively, representing 9, 10, 14-fold increases relative to the TAT-free control conjugate, [89Zr]Zr-DFO-Her-TAT(0).

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

Alcohol-related emergency department attendances after the introduction of the Sale and Supply of Alcohol Act 2012.

AIM: To measure changes in alcohol-related emergency department (ED) attendances after introduction of the Sale and Supply of Alcohol Act 2012. METHODS: Cross-sectional survey of Christchurch ED attendees in three-week sampling periods in 2013 and 2017. Participants had consumed alcohol within four hours, or their drinking had directly contributed to the attendance. The quantity of alcohol consumed and places of purchase and consumption for the index drinking episode were recorded. RESULTS: From 2013 to 2017 there was a non-significant (p=.41) reduction in the proportion of ED attendees eligible for the study, from 253/3400 (7.4%) to 258/3721 (6.9%). Among participants (n=169 in 2013, n=139 in 2017), liquor store purchasing increased from 41.7% in 2013 to 56.1% in 2017 (p<.01) but there was no significant change in quantity consumed in the index episode; last drink location; percentage of participants with an injury-related attendance; or pre-drinking. In both waves, most participants had purchased alcohol from off-licence venues and consumed their last drink at a private location. CONCLUSION: Alcohol-related ED attendances remained common after the Sale and Supply of Alcohol Act 2012 was introduced, and they mainly occurred in people who sourced alcohol from off-licence outlets and had their last drink at private locations.

Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity.

Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically plausible (including unnatural but functionally relevant) side chains is not readily accessible. We describe C (sp3)-C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free-radical chemistry, and use them to form Cß-Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide diversity of natural, unnatural, posttranslationally modified (methylated, glycosylated, phosphorylated, hydroxylated), and labeled (fluorinated, isotopically labeled) side chains to be added to a common, readily accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for access to diverse proteins.