Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 μM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/β and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor.

The proinflammatory response leads to various types of pathologic pathways, including the development of preterm birth. Preterm birth occurs in 12% of deliveries in the United States and causes more than 70% of perinatal morbidity and mortality. The most common cause of spontaneous preterm birth is intrauterine infection in the mother. There is accumulating evidence indicating that the release of proinflammatory cytokines plays a critical role in the pathogenesis of inflammation-associated premature delivery. We found that the common organic solvent, N,N-dimethylacetamide (DMA), prevents endotoxin-induced preterm birth in timed pregnant C57BL/6 embryonic day (E)15.5 mice and rescues their pups from spontaneous abortion at doses many-fold lower than those currently used clinically and in a dose-dependent fashion. We also provide histologic evidence that DMA suppresses the endotoxin-triggered proinflammatory response by significantly attenuating inflammatory cell infiltration of placental tissue. Furthermore, immunoblotting analysis of placental tissue harvested from our murine models revealed DMA-mediated regulation of expression of the proinflammatory cytokines IL-1β, tumor necrosis factor α, and IL-6, and increased expression of the regulatory inflammatory cytokine IL-10. By using in vitro studies, we provide evidence that DMA suppresses macrophage function and that this small molecule prevents nuclear translocation of nuclear factor-kB. These results suggest that DMA represents a newly discovered, nontoxic therapy for a broad range of inflammatory disorders.

Aim To investigate the association between vasculopathy and survival during experimental cerebral malaria (ECM), and to determine whether targeting the endothelin-1 (ET-1) pathway alone or in combination with the anti-malaria drug artemether (a semi-synthetic derivative of artemisinin) will improve microvascular hemorrhage and survival. Main Methods C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) were randomly assigned to four groups: no treatment, artemether treated, ETA receptor antagonist (HJP-272) treated, or HJP-272 and artemether treated. The uninfected control mice were treated with HJP-272 and artemether. We analyzed survival, cerebral hemorrhage, weight change, blood glucose levels and parasitemia. Key Findings Our studies demonstrated decreased brain hemorrhage in PbA-infected (ECM) mice treated when HJP-272, a 1,3,6-trisubstituted-2-carboxy-quinol-4-one novel ETA receptor antagonist synthesized by our group, is used in conjunction with artemether, an anti-malarial agent. In addition, despite adversely affecting parasitemia and weight in non-artemether treated infected mice, HJP-272, seemed to confer some survival benefit when used as adjunctive therapy, though this did not reach significance. Significance Previous studies demonstrate that the endothelin pathway is associated with vasculopathy, neuronal injury and inflammation in ECM. As demonstrated here, components of the ET-1 pathway may be important targets for adjunctive therapy in ECM, and may help in preventing hemorrhage and in improving survival when used as adjunctive therapy during malaria infection. The data presented suggest that our novel agent, HJP-272, may ameliorate alterations in the vasculature which can potentially lead to inflammation, neurological dysfunction, and subsequent death in mice with ECM.

Here, we report for the first time, PD173074, an inhibitor of the FGFR, to selectively reverse ABCB1 transporter-mediated MDR by directly blocking the efflux function of the transporter.