Activity-directed synthesis (ADS), a novel discovery approach in which bioactive molecules emerge in parallel with associated syntheses, was exploited to develop a weakly binding fragment into novel androgen receptor agonists. Harnessing promiscuous intermolecular reactions of carbenoid compounds enabled highly efficient exploration of chemical space. Four substrates were prepared, yet exploited in 326 reactions to explore diverse chemical space; guided by bioactivity alone, the products of just nine of the reactions were purified to reveal diverse novel agonists with up to 125-fold improved activity. Remarkably, one agonist stemmed from a novel enantioselective transformation; this is the first time that an asymmetric reaction has been discovered solely on the basis of the biological activity of the product. It was shown that ADS is a significant addition to the lead generation toolkit, enabling the efficient and rapid discovery of novel, yet synthetically accessible, bioactive chemotypes.

Activity-directed synthesis (ADS), a novel discovery approach in which bioactive molecules emerge in parallel with associated syntheses, was exploited to develop a weakly binding fragment into novel androgen receptor agonists. Harnessing promiscuous intermolecular reactions of carbenoid compounds enabled highly efficient exploration of chemical space. Four substrates were prepared, yet exploited in 326 reactions to explore diverse chemical space; guided by bioactivity alone, the products of just nine of the reactions were purified to reveal diverse novel agonists with up to 125-fold improved activity. Remarkably, one agonist stemmed from a novel enantioselective transformation; this is the first time that an asymmetric reaction has been discovered solely on the basis of the biological activity of the product. It was shown that ADS is a significant addition to the lead generation toolkit, enabling the efficient and rapid discovery of novel, yet synthetically accessible, bioactive chemotypes.

Despite the importance of post-translational modifications in controlling the solubility and conformational properties of proteins and peptides, precisely how the aggregation propensity of different peptide sequences is modulated by chemical modification remains unclear. Here we have investigated the effect of phosphorylation on the aggregation propensity of a 13-residue synthetic peptide incorporating one or more phosphate groups at seven different sites at various pH values. Fibril formation was shown to be inhibited when a single phosphate group was introduced at all seven locations in the peptide sequence at pH 7.5, when the phosphate group is fully charged. By contrast, when the same peptides were analysed at pH 1.1, when the phosphate is fully protonated, fibrils from all seven peptide sequences form rapidly. At intermediate pH values (pH 3.6) when the phosphate group is mono-anionic, the aggregation propensity of the peptides was found to be highly dependent on the position of the phosphate group in the peptide sequence. Using this information, combined with molecular dynamics (MD) simulations of the peptide sequence, we provide evidence consistent with the peptide forming amyloid fibrils with a class 7 architecture. The results highlight the potential utility of phosphorylation as a method of reversibly controlling the aggregation kinetics of peptide sequences both during and after synthesis. Moreover, by exploiting the ability of the phosphate group to adopt different charge states as a function of pH, and combining experimental insights with atomistic information calculated from MD simulations as pH is varied, we show how the resulting information can be used to predict fibril structures consistent with both datasets, and use these to rationalise their sensitivity of fibrillation kinetics both to the location of the phosphate group and its charge state.