A network of molecular chaperones is known to bind proteins (“clients”) and balance their folding, function and turnover. However, it is often not clear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein aggregation diseases. In this study, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of significance, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease. Impact Statement Large-scale screening of chaperone interactions with tau and its variants identified DnaJA2 as a key protective factor in tauopathy.

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored solubility in aged mouse and human lenses. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.

Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor. In contrast to cotransin, apratoxin A does not exhibit a substrate-selective inhibitory mechanism, but blocks ER translocation of all tested Sec61 clients with similar potency. Our results suggest that multiple structurally unrelated natural products have evolved to target overlapping but non-identical binding sites on Sec61, thereby producing distinct biological outcomes.

Heat shock protein 70 (Hsp70) is a stress-inducible molecular chaperone that is required for cancer development at several steps. Targeting the active site of Hsp70 has proven relatively challenging, driving interest in alternative approaches. Hsp70 collaborates with the Bcl2-associated athanogene 3 (Bag3) to promote cell survival through multiple pathways, including FoxM1. Therefore, inhibitors of the Hsp70-Bag3 protein-protein interaction (PPI) may provide a non-canonical way to target this chaperone. We report that JG-98, an allosteric inhibitor of this PPI, indeed has anti-proliferative activity (EC50 values between 0.3 and 4 μM) across cancer cell lines from multiple origins. JG-98 destabilized FoxM1 and relieved suppression of downstream effectors, including p21 and p27. Based on these findings, JG-98 was evaluated in mice for pharmacokinetics, tolerability and activity in two xenograft models. The results suggested that the Hsp70-Bag3 interaction may be a promising, new target for anti-cancer therapy.

Peptidyl-proline isomerases (PPIases) are a chaperone superfamily comprising the FK506-binding proteins (FKBPs), cyclophilins, and parvulins. PPIases catalyze the cis/trans isomerization of proline, acting as a regulatory switch during folding, activation, and/or degradation of many proteins. These “clients” include proteins with key roles in cancer, neurodegeneration, and psychiatric disorders, suggesting that PPIase inhibitors could be important therapeutics. However, the active site of PPIases is shallow, solvent-exposed, and well conserved between family members, making selective inhibitor design challenging. Despite these hurdles, macrocyclic natural products, including FK506, rapamycin, and cyclosporin, bind PPIases with nanomolar or better affinity. De novo attempts to derive new classes of inhibitors have been somewhat less successful, often showcasing the “undruggable” features of PPIases. Interestingly, the most potent of these next-generation molecules tend to integrate features of the natural products, including macrocyclization or proline mimicry strategies. Here, we review recent developments and ongoing challenges in the inhibition of PPIases, with a focus on how natural products might inform the creation of potent and selective inhibitors.