Background and purpose: Inhibition of hepatic glycogen phosphorylase is a potential treatment for glycaemic control in type 2 diabetes. Selective inhibition of the liver phosphorylase isoform could minimize adverse effects in other tissues. We investigated the potential selectivity of two indole site phosphorylase inhibitors, GPi688 and GPi819. Experimental approach: The activity of glycogen phosphorylase was modulated using the allosteric effectors glucose or caffeine to promote the less active T state, and AMP to promote the more active R state. In vitro potency of indole site inhibitors against liver and muscle glycogen phosphorylase a was examined at different effector concentrations using purified recombinant enzymes. The potency of GPi819 was compared with its in vivo efficacy at raising glycogen concentrations in liver and muscle of Zucker (fa/fa) rats. Key results: In vitro potency of indole site inhibitors depended upon the activity state of phosphorylase a. Both inhibitors showed selectivity for liver phosphorylase a when the isoform specific activities were equal. After 5 days dosing of GPi819 (37.5 mmol kg À1 ), where free compound levels in plasma and tissue were at steady state, glycogen elevation was 1.5-fold greater in soleus muscle than in liver (Po0.05). Conclusions and implications:The in vivo selectivity of GPi819 did not match that seen in vitro when the specific activities of phosphorylase a isoforms are equal. This suggests T state promoters may be important physiological regulators in skeletal muscle. The greater efficacy of indole site inhibitors in skeletal muscle has implications for the overall safety profile of such drugs.

USP7 (HAUSP) is a deubiquitinating enzyme, which plays a crucial role in regulating the levels of the p53 tumour suppressor protein, through its ability to prevent the proteasomal degradation of the Ubiquitin ligase for p53, Hdm2. Supporting evidence suggests that an inhibitor of USP7 would act to abrogate the action of Hdm2, and thereby elevate levels of the p53 protein, with associated therapeutic benefits in cancer and potentially other diseases. In this article, we describe the characterisation of differential enzyme activity of both the full length and putative catalytic domain of human USP7 expressed in both bacterial and insect cell expression systems. We also demonstrate the way in which variations in the reducing environment surrounding the enzyme can dramatically affect both the stability of the enzyme and the range of small molecules able to inhibit the catalytic activity of the enzyme. Furthermore, we describe the validation and use of this assay for a high-throughput screening approach, again highlighting the critical nature of the enzyme's environment. Taken together, these findings not only increase our understanding of the enzymatic activity of deubiquitinating enzymes, but also highlight several key considerations of importance in the development of therapeutic agents against this novel class of therapeutic targets.

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BackgroundThere are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N.MethodsWe used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe.ResultsBinding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased.ConclusionsThe results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.