Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables an understanding of structural features directing affinity and selectivity in the active site. Here, we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key molecular features of inhibitor linker and capping groups that facilitate and differentiate binding to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft defined by F583 and F643, and different aromatic linkers direct the capping group toward shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets. These results expand our understanding of factors contributing to the selective inhibition of HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.

Isoprenoids are vital to all organisms in supporting core functions of life, like respiration and membrane stability. 1 IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential to gram-negative bacteria, mycobacteria and apicomplexans. 2 , 3 The IspH substrate, HMBPP, is not produced in humans and other metazoans and activates cytotoxic Vγ9Vδ2 T-cells in humans and primates at extremely low concentrations. 4 - 6 We describe novel IspH inhibitors and through structure-guided analog design, refine their potency to nanomolar levels. We have modified these into prodrugs for delivery into bacteria and report that they kill clinical isolates of several multidrug resistant bacterial species such as Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia , Mycobacterium and Bacillus , while being relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with prodrugs resemble those with conditional IspH knockdown. Notably, these prodrugs also cause expansion and activation of human Vγ9Vδ2 T-cells in a humanized mouse model of bacterial infection. These IspH prodrugs synergize direct antibiotic killing with a simultaneous rapid immune response by cytotoxic γδ T-cells, which may limit the rise of antibiotic resistant bacterial populations.

The protein chaperone HSP70 is overexpressed in many cancers including colorectal cancer, where overexpression is associated with poor survival. We report here the creation of a uniquely acting HSP70 inhibitor (HSP70i) that targets multiple compartments in the cancer cell, including mitochondria. This inhibitor was mitochondria toxic and cytotoxic to colorectal cancer cells, but not to normal colon epithelial cells. Inhibition of HSP70 was efficacious as a single agent in primary and metastatic models of colorectal cancer and enabled identification of novel mitochondrial client proteins for HSP70. In a syngeneic colorectal cancer model, the inhibitor increased immune cell recruitment into tumors. Cells treated with the inhibitor secreted danger-associated molecular patterns (DAMP), including ATP and HMGB1, and functioned effectively as a tumor vaccine. Interestingly, the unique properties of this HSP70i in the disruption of mitochondrial function and the inhibition of proteostasis both contributed to DAMP release. This HSP70i constitutes a promising therapeutic opportunity in colorectal cancer and may exhibit antitumor activity against other tumor types. Significance: These findings describe a novel HSP70i that disrupts mitochondrial proteostasis, demonstrating single-agent efficacy that induces immunogenic cell death in treated tumors.

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The South American social spider Parawixia bistriata produces a venom containing complex organic compounds with intriguing biological activities. The crude venom leads to paralysis in termites and stimulates l-glutamate uptake and inhibits GABA uptake in rat brain synaptosomes. Glutamate is the major neurotransmitter at the insect neuromuscular junction and at the mammalian central nervous system, suggesting a modulation of the glutamatergic system by the venom. Parawixin1, 2, and 10 (Pwx1, 2 and 10) are HPLC fractions that demonstrate this bioactivity. Pwx1 stimulates l-glutamate uptake through the main transporter in the brain, EAAT2, and is neuroprotective in in vivo glaucoma models. Pxw2 inhibits GABA and glycine uptake in synaptosomes and inhibits seizures and neurodegeneration, and Pwx10 increases l-glutamate uptake in synaptosomes and is neuroprotective and anticonvulsant, shown in in vivo epilepsy models. Herein, we investigated the low molecular mass compounds in this venom and have found over 20 small compounds and 36 unique acylpolyamines with and without amino acid linkers. The active substances in fractions Pwx1 and Pwx2 require further investigation. We elucidated and confirmed the structure of the active acylpolyamine in Pwx10. Both fraction Pwx10 and the synthesized component enhance the activity of transporters EAAT1 and EAAT2, and, importantly, offer in vitro neuroprotection against excitotoxicity in primary cultures. These data suggest that compounds with this mechanism could be developed into therapies for disorders in which l-glutamate excitotoxicity is involved.