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SummaryMethane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilisation of methane through anaerobic methane oxidation. The enzyme employs an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a novel methyl radical/Ni(II)-thiolate intermediate. However, the biosynthesis of coenzyme F430 from the common primogenitor uroporphyrinoge III, incorporating 11 steric centres into the macrocycle, has remained poorly understood although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamisation and carbocyclic ring formation. We have now identified the proteins that catalyse coenzyme F430 biosynthesis from sirohydrochlorin, termed CfbA-E, and shown their activity. The research completes our understanding of how nature is able to construct its repertoire of tetrapyrrole-based life pigments, permitting the development of recombinant systems to utilise these metalloprosthetic groups more widely.

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Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/β-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADp-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and costeffective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein-protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions. Poly(ADP-ribosyl) polymerases (PARPs) called tankyrases (TNKSs) are key regulators of diverse cellular processes such as mitotic spindle formation, telomere homeostasis, Wnt/β-catenin signalling and glucose metabolism 1-5. In humans, two tankyrases with overlapping functions exist and are termed TNKS1 and TNKS2 6,7. Like other enzymes of the PARP family, tankyrases catalyse the transfer of multiple ADP-ribose units to their protein substrates 8-10 , thus leaving them poly-ADP-ribosylated. In many cases this serves as a signal for subsequent ubiquitination and thereby proteasomal degradation 11,12. Prominent targets of tankyrases include Axin1/2 13,14 , a major regulator of β-catenin levels, TRF1 7,9,15,16 , a telomere binding protein that inhibits telomere extension and NuMA 17-19 , a protein involved in the formation of spindle-poles during mitosis. The major role of tankyrases in the regulation of β-catenin levels has led to the development of multiple TNKS inhibitors 2,20-22. These inhibitors function by binding to the NAD + binding pocket of the catalytic ARTD domain (ADP-ribosyl-transferases diphtheria toxin-like), inhibiting the poly-ADP-ribosylation function. However, it has been shown that tankyrases also contribute through oligomerization and mediation of protein-protein interactions to the Wnt-signalling pathway 23. We refer to these non-catalytic functions of tankyrases as scaffolding functions. By being able to specifically target different domains involved in the scaffolding function with a tool compound, it could be possible to inves...

Tankyrases 1 and 2 are central biotargets in the WNT/β-catenin signaling and Hippo signaling pathways. We have previously developed tankyrase inhibitors bearing a 1,2,4-triazole moiety and binding predominantly to the adenosine binding site of the tankyrase catalytic domain. Here we describe a systematic structure-guided lead optimization approach of these tankyrase inhibitors. The central 1,2,4-triazole template and trans- cyclobutyl linker of the lead compound 1 were left unchanged, while side-group East, West, and South moieties were altered by introducing different building blocks defined as point mutations. The systematic study provided a novel series of compounds reaching picomolar IC 50 inhibition in WNT/β - catenin signaling cellular reporter assay. The novel optimized lead 13 resolves previous atropisomerism, solubility, and Caco-2 efflux liabilities. 13 shows a favorable ADME profile, including improved Caco-2 permeability and oral bioavailability in mice, and exhibits antiproliferative efficacy in the colon cancer cell line COLO 320DM in vitro.