Biflavonoid-Induced Disruption of Hydrogen Bonds Leads to Amyloid-ß Disaggregation.

Deposition of amyloid ß (Aß) fibrils in the brain is a key pathologic hallmark of Alzheimer's disease. A class of polyphenolic biflavonoids is known to have anti-amyloidogenic effects by inhibiting aggregation of Aß and promoting disaggregation of Aß fibrils. In the present study, we further sought to investigate the structural basis of the Aß disaggregating activity of biflavonoids and their interactions at the atomic level. A thioflavin T (ThT) fluorescence assay revealed that amentoflavone-type biflavonoids promote disaggregation of Aß fibrils with varying potency due to specific structural differences. The computational analysis herein provides the first atomistic details for the mechanism of Aß disaggregation by biflavonoids. Molecular docking analysis showed that biflavonoids preferentially bind to the aromatic-rich, partially ordered N-termini of Aß fibril via the π-π interactions. Moreover, docking scores correlate well with the ThT EC50 values. Molecular dynamic simulations revealed that biflavonoids decrease the content of ß-sheet in Aß fibril in a structure-dependent manner. Hydrogen bond analysis further supported that the substitution of hydroxyl groups capable of hydrogen bond formation at two positions on the biflavonoid scaffold leads to significantly disaggregation of Aß fibrils. Taken together, our data indicate that biflavonoids promote disaggregation of Aß fibrils due to their ability to disrupt the fibril structure, suggesting biflavonoids as a lead class of compounds to develop a therapeutic agent for Alzheimer's disease.

Novel Noncompetitive Type Three Secretion System ATPase Inhibitors Shut Down Shigella Effector Secretion.

Shigella is the causative agent of bacillary dysentery and is responsible for an estimated 165 million infections and 600,000 deaths annually. Like many Gram-negative pathogens, Shigella relies on a type three secretion system (T3SS) to initiate and sustain infection by directly injecting effector proteins into host cells. Protein secretion through the needle-like injectisome and overall Shigella virulence rely on the T3SS ATPase Spa47, making it a likely means for T3SS regulation and an attractive target for therapeutic small molecule inhibitors. Here, we utilize a recently solved 2.15 Å crystal structure of Spa47 to computationally screen 7.6 million drug-like compounds for candidates which avoid the highly conserved active site by targeting a distal, but critical, interface between adjacent protomers of the Spa47 homohexamer. Ten of the top inhibitor candidates were characterized, identifying novel Spa47 inhibitors that reduce in vitro ATPase activity by as much as 87.9 ± 10.5% with IC50's as low as 25 ± 20 µM and reduce in vivo Shigella T3SS protein secretion by as much as 94.7 ± 3.0%. Kinetic analyses show that the inhibitors operate through a noncompetitive mechanism that likely supports the inhibitors' low cytotoxicity, as they avoid off-target ATPases involved in either Shigella or mammalian cell metabolism. Interestingly, the inhibitors display nearly identical inhibition profiles for Spa47 and the T3SS ATPases EscN from E. coli and FliI from Salmonella. Together, the results of this study provide much-needed insight into T3SS ATPase inhibition mechanisms and a strong platform for developing broadly effective cross-pathogen T3SS ATPase inhibitors.

Shutting Down Shigella Secretion: Characterizing Small Molecule Type Three Secretion System ATPase Inhibitors.

Many important human pathogens rely on one or more type three secretion systems (T3SSs) to inject bacterial effector proteins directly into the host cell cytoplasm. Secretion of protein through the needlelike type three secretion apparatus (T3SA) is essential for pathogen virulence and relies on a highly conserved ATPase at the base of the apparatus, making it an attractive target for anti-infective therapeutics. Here, we leveraged the ability to purify an active oligomeric Shigella T3SS ATPase to provide kinetic analyses of three T3SS ATPase inhibitors of Spa47. In agreement with in silico docking simulations, the inhibitors displayed noncompetitive inhibition profiles and efficiently reduced Spa47 ATPase activity with IC50s as low as 52 ± 3 µM. Two of the inhibitors functioned well in vivo, nearly abolishing effector protein secretion without significantly affecting the Shigella growth phenotype or HeLa cell viability. Furthermore, characterization of Spa47 complexes in vitro and Shigella T3SA formation in vivo showed that the inhibitors do not function through disruption of Spa47 oligomers or by preventing T3SA formation. Together, these findings suggest that inhibitors targeting Spa47 may be an effective means of combating Shigella infection by shutting down type three secretion without preventing presentation of the highly antigenic T3SA tip proteins that aid in clearing the infection and developing pan- Shigella immunological memory. In summary, this is the first report of Shigella T3SS ATPase inhibitors and one of only a small number of studies characterizing T3SS ATPase inhibition in general. The work presented here provides much-needed insight into T3SS ATPase inhibition mechanisms and provides a strong platform for developing and evaluating non-antibiotic therapeutics targeting Spa47 and other T3SS ATPases.