A first-in-class inhibitor of Hsp110 molecular chaperones of pathogenic fungi.

Proteins of the Hsp110 family are molecular chaperones that play important roles in protein homeostasis in eukaryotes. The pathogenic fungus Candida albicans, which causes infections in humans, has a single Hsp110, termed Msi3. Here, we provide proof-of-principle evidence supporting fungal Hsp110s as targets for the development of new antifungal drugs. We identify a pyrazolo[3,4-b] pyridine derivative, termed HLQ2H (or 2H), that inhibits the biochemical and chaperone activities of Msi3, as well as the growth and viability of C. albicans. Moreover, the fungicidal activity of 2H correlates with its inhibition of in vivo protein folding. We propose 2H and related compounds as promising leads for development of new antifungals and as pharmacological tools for the study of the molecular mechanisms and functions of Hsp110s.

Magnolol-loaded cholesteryl biguanide conjugate hydrochloride nanoparticles for triple-negative breast cancer therapy.

The potential of combination therapy using nanoparticle delivery systems in improving triple-negative breast cancer treatment efficacy remains to be explored. Here, we report a novel nanoparticle system using a cholesterol biguanide conjugate hydrochloride (CBH) as both a drug and carrier to load magnolol (MAG). Poly(ethylene glycol)-poly(lactic-co-glycolic acid) (mPEG-PLGA) and aminoethyl anisamide-poly(ethylene glycol)-poly(lactic-co-glycolic acid) (AEAA-PEG-PLGA) were added to form nanoparticles. Nanoparticles accumulated most in tumor tissues when the weight ratio of AEAA-PEG-PLGA to mPEG-PLGA was 4:1. MAG and CBH exerted a synergistic inhibitory effect on 4 T1 cells. An in vitro study showed that nanoparticles displayed the highest tumor cell uptake rate, highest apoptosis rate, and strongest inhibitory effect on tumor cell migration and monoclonal formation. CBH might promote nanoparticle uptake by cells and lysosomal escape. After intravenous administration to mice with 4 T1 breast tumors in situ, the nanoparticles inhibited tumor growth without obvious toxicity. Western blot results showed that nanoparticles altered the levels of p53, p-AKT, and p-AMPK in the tumor tissue. Moreover, cell apoptosis was found in the same area of H&E-stained and TUNEL-stained tumors treated with the nanoparticles. Collectively, this nanoparticle system provides a novel combination drug delivery strategy for treating triple-negative breast cancer.

Purification and biochemical characterization of Msi3, an essential Hsp110 molecular chaperone in Candida albicans.

Hsp110s are unique and essential molecular chaperones in the eukaryotic cytosol. They play important roles in maintaining cellular protein homeostasis. Candida albicans is the most prevalent yeast opportunistic pathogen that causes fungal infections in humans. As the only Hsp110 in Candida albicans, Msi3 is essential for the growth and infection of Candida albicans. In this study, we have expressed and purified Msi3 in nucleotide-free state and carried out biochemical analyses. Sse1 is the major Hsp110 in budding yeast S. cerevisiae and the best characterized Hsp110. Msi3 can substitute Sse1 in complementing the temperature-sensitive phenotype of S. cerevisiae carrying a deletion of SSE1 gene although Msi3 shares only 63.4% sequence identity with Sse1. Consistent with this functional similarity, the purified Msi3 protein shares many similar biochemical activities with Sse1 including binding ATP with high affinity, changing conformation upon ATP binding, stimulating the nucleotide-exchange for Hsp70, preventing protein aggregation, and assisting Hsp70 in refolding denatured luciferase. These biochemical characterizations suggested that Msi3 can be used as a model for studying the molecular mechanisms of Hsp110s.

Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability.

Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter-repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.