NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90.

The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90ß, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.

Structures of Hsp90α and Hsp90ß bound to a purine-scaffold inhibitor reveal an exploitable residue for drug selectivity.

Hsp90α and Hsp90ß are implicated in a number of cancers and neurodegenerative disorders but the lack of selective pharmacological probes confounds efforts to identify their individual roles. Here, we analyzed the binding of an Hsp90α-selective PU compound, PU-11-trans, to the two cytosolic paralogs. We determined the co-crystal structures of Hsp90α and Hsp90ß bound to PU-11-trans, as well as the structure of the apo Hsp90ß NTD. The two inhibitor-bound structures reveal that Ser52, a nonconserved residue in the ATP binding pocket in Hsp90α, provides additional stability to PU-11-trans through a water-mediated hydrogen-bonding network. Mutation of Ser52 to alanine, as found in Hsp90ß, alters the dissociation constant of Hsp90α for PU-11-trans to match that of Hsp90ß. Our results provide a structural explanation for the binding preference of PU inhibitors for Hsp90α and demonstrate that the single nonconserved residue in the ATP-binding pocket may be exploited for α/ß selectivity.

Selective GSK3ß Inhibition Mediates an Nrf2-Independent Anti-inflammatory Microglial Response.

Glycogen synthase kinase 3 (GSK3) is associated with the proinflammatory phenotype of microglia and has been shown to act in concert with nuclear factor kappa B (NF-κB). GSK3 is also a suppressor of nuclear factor erythroid 2-related factor 2 (Nrf2), the principal regulator of redox homeostasis. Agreeing with the oxidative paradigm of aging, Nrf2 is often deregulated in parainflammatory and neurodegenerative diseases. In this study, we aimed to explore a multimodal disease-modifying utility of GSK3 inhibition, beyond neuronal proteopathologies. Furthermore, we aimed to underscore the difference in therapeutic value between the two GSK3 paralogs by isoform-selective chemical inhibition. The anti-inflammatory effects of paralog-selective GSK3 inhibitors were evaluated as a function of the reductive capacity of each to mitigate LPS-induced activation of SIM-A9 microglia. The Griess method was employed to detect the nitrate-lowering capacity of selective GSK3 inhibition. Real-time PCR was used to assess post-treatment expression levels of pro-inflammatory markers and antioxidant genes; pro-inflammatory cytokines were assayed by ELISA. Nuclear lysates of treated cells were examined for Nrf2 and NF-κB accumulation by immunoblotting. Finally, to infer whether the counter-inflammatory activity of GSK3 inhibition was Nrf2-dependent, DsiRNA-mediated knockdown of Nrf2 was attempted. Results from our experiments reveal a superior anti-inflammatory and anti-oxidative efficacy for GSK3ß-selective inhibition, compared to GSK3α-selective and non-selective pan-inhibition; hence, use of selective GSK3ß inhibitors is likely to be more propitious than non-selective dual inhibitors administered at comparable doses. Moreover, our results suggest that the anti-inflammatory effects of GSK3 inhibition are not Nrf2 dependent.