Activation of the PERK branch of the unfolded protein response (UPR) in response to protein misfolding within the endoplasmic reticulum (ER) results in the transient repression of protein synthesis, mediated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This is part of a wider integrated physiological response to maintain proteostasis in the face of ER stress, the dysregulation of which is increasingly associated with a wide range of diseases, particularly neurodegenerative disorders. In prion-diseased mice, persistently high levels of eIF2α cause sustained translational repression leading to catastrophic reduction of critical proteins, resulting in synaptic failure and neuronal loss. We previously showed that restoration of global protein synthesis using the PERK inhibitor GSK2606414 was profoundly neuroprotective, preventing clinical disease in prion-infected mice. However, this occured at the cost of toxicity to secretory tissue, where UPR activation is essential to healthy functioning. Here we show that pharmacological modulation of eIF2α-P-mediated translational inhibition can be achieved to produce neuroprotection without pancreatic toxicity. We found that treatment with the small molecule ISRIB, which restores translation downstream of eIF2α, conferred neuroprotection in prion-diseased mice without adverse effects on the pancreas. Critically, ISRIB treatment resulted in only partial restoration of global translation rates, as compared with the complete restoration of protein synthesis seen with GSK2606414. ISRIB likely provides sufficient rates of protein synthesis for neuronal survival, while allowing some residual protective UPR function in secretory tissue. Thus, fine-tuning the extent of UPR inhibition and subsequent translational de-repression uncouples neuroprotective effects from pancreatic toxicity. The data support the pursuit of this approach to develop new treatments for a range of neurodegenerative disorders that are currently incurable.

Inhibition of the ATPase activity of the chaperone protein HSP90 is a potential strategy for treatment of cancers. We have determined structures of the HSP90alpha N-terminal domain complexed with the purine-based inhibitor, PU3, and analogs with enhanced potency both in enzyme and cell-based assays. The compounds induce upregulation of HSP70 and downregulation of the known HSP90 client proteins Raf-1, CDK4, and ErbB2, confirming that the molecules inhibit cell growth by a mechanism dependent on HSP90 inhibition. We have also determined the first structure of the N-terminal domain of HSP90beta, complexed with PU3. The structures allow a detailed rationale to be developed for the observed affinity of the PU3 class of compounds for HSP90 and also provide a structural framework for design of compounds with improved binding affinity and drug-like properties.

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the β and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.