Rapamycin-inspired macrocycles with new target specificity

Rapamycin and FK506 are macrocyclic natural products with an extraordinary mode of action, in which they form binary complexes with FK506-binding protein (FKBP) through a shared FKBP-binding domain before forming ternary complexes with their respective targets, mechanistic target of rapamycin (mTOR) and calcineurin, respectively. Inspired by this, we sought to build a rapamycin-like macromolecule library to target new cellular proteins by replacing the effector domain of rapamycin with a combinatorial library of oligopeptides. We developed a robust macrocyclization method using ring-closing metathesis and synthesized a 45,000-compound library of hybrid macrocycles (named rapafucins) using optimized FKBP-binding domains. Screening of the rapafucin library in human cells led to the discovery of rapadocin, an inhibitor of nucleoside uptake. Rapadocin is a potent, isoform-specific and FKBP-dependent inhibitor of the equilibrative nucleoside transporter 1 and is efficacious in an animal model of kidney ischaemia reperfusion injury. Together, these results demonstrate that rapafucins are a new class of chemical probes and drug leads that can expand the repertoire of protein targets well beyond mTOR and calcineurin.Peer reviewe

Phosphorus availability in chicken manure is lower with increased stockpiling period, despite a larger orthophosphate content

Background and aims: The relative proportions of phosphorus (P) forms present in manure will determine the overall availability of manure P to plants; however, the link between the forms of P in manures and manure P availability is unclear. This study compares the bioavailability and P speciation of three manures of different stockpiling duration: less than 1 month, 6 months and 12 months; manures were collected concurrently from a single poultry farm. Methods: Bioavailability to wheat in a glasshouse trial was measured using an isotopic dilution method with manure added at an application rate equivalent to 20 kg P ha-1. Phosphorus speciation was measured by 31P nuclear magnetic resonance (NMR) spectroscopic analysis of NaOH-EDTA extracts of the manures. Results: The addition of all manures significantly increased shoot biomass and P concentration, with the fresh manure having the greatest effect. Addition of the fresh manure resulted in the largest labile P pool, highest manure P uptake and manure P recovery, while the manure stockpiled for 12 months resulted in the lowest manure P uptake and manure P recovery. NMR analysis indicated that there was more monoester organic P, especially phytate, in manure stockpiled for shorter periods, while the proportion of manure P that was orthophosphate increased with stockpiling time. Conclusions: Together, these results imply that although the proportion of total P in the manures detected as orthophosphate was higher with longer stockpiling, only a fraction of this orthophosphate was plant-available. This suggests the availability of P from orthophosphate in manures decreases with longer stockpiling time in much the same way that P from orthophosphate in mineral fertilizer becomes less available in soil over time. © 2013 Springer Science+Business Media Dordrecht.C. A. E. Peirce, R. J. Smernik, T. M. McBeat