MF-094

MF-094 nanodelivery inhibits oral squamous cell carcinoma by targeting USP30

Background: Oral squamous cell carcinoma (OSCC) is a common head and neck cancer, and the incidence of OSCC is increasing. As the mortality of OSCC keeps increasing, it is crucial to clarify its pathogenesis and develop new therapeutic strategies. Methods: Confocal laser scanning microscopy was used to evaluate the uptake of nanoparticles (NPs). The potential functions of USP30 were evaluated by cell counting kit (CCK)-8, flow cytometry, biochemical assay, coimmunoprecipitation, qRT-PCR, and immunoblotting. The antitumor effect of NP-loaded USP30 inhibitor MF-094 was evaluated in vitro and in vivo. Results: In this study, increased USP30 expression was found in OSCC specimens and cell lines through qRT-PCR and immunoblotting. CCK-8, flow cytometry, and biochemical assay revealed that the deubiquitylated catalytic activity of USP30 contributed to cell viability and glutamine consumption of OSCC. Subsequently, USP30 inhibitor MF-094 was loaded in ZIF-8-PDA and PEGTK to fabricate ZIF-8-PDA-PEGTK nanoparticles, which exhibited excellent inhibition of cell viability and glutamine consumption of OSCC, both in vitro and in vivo. Conclusion: The results indicated the clinical significance of USP30 and showed that nanocomposites provide a targeted drug delivery system for treating OSCC.

MF-094, a potent and selective USP30 inhibitor, accelerates diabetic wound healing by inhibiting the NLRP3 inflammasome

Diabetes is a prevalent disease worldwide that can result in several complications, including renal failure, blindness, and amputation. Diabetic foot ulcers, which have the characteristics of chronic wounds, are a devastating component of diabetes progression that can lead to lower extremity amputation. In this study, we set out to investigate the mechanisms involved in wound healing of diabetic foot ulcers. The expression of USP30 in skin tissues of patients with diabetic foot ulcers and HSF2 human skin fibroblasts treated with advanced glycation end (AGE) products was detected by qRT-PCR, and CCK-8, cell scratch and ELISA assay were used to detect cell viability, migration and levels of Col I, Col III, MMP-2, MMP-9, IL-1β and IL-18. The interaction between USP30 and NLRP3 was verified by co-immunoprecipitation and ubiquitination assays. The expression of USP30, NLRP3 and caspase-1 p20 was detected by Western blot. USP30 inhibitor MF-094 was used to treat diabetic rat model established by streptozotocin (STZ). We found that USP30, a deubiquitinase, was upregulated in skin tissues of patients with diabetic foot ulcers compared with normal skin tissues. In vitro, we found that treatment of HSF2 human skin fibroblasts with advanced glycation end (AGE) products, known to contribute to diabetic complications, resulted in suppressed viability and migration of HSF2 cells, as well as increased levels of USP30 mRNA and protein. Functionally, downregulation of USP30 via shRNA-mediated knockdown or treatment with the USP30 inhibitor MF-094, restored viability and migration of AGE-treated HSF2 cells. We identified the NLRP3 inflammasome as a critical target of USP30 in AGE-induced functions. Mechanistically, we demonstrate that USP30 activates the NLRP3 inflammasome by deubiquitinating NLRP3. Finally, we show that inhibition of USP30 via MF-094 treatment facilitated wound healing in diabetic rats and resulted in decreased protein levels of NLRP3 and its downstream target caspase-1 p20, thus establishing the physiological importance of the identified USP30-NLRP3 link. Together, our findings suggest a therapeutic potential for USP30 in diabetic foot ulcers.

Increased FUN14 domain containing 1 (FUNDC1) ubiquitination level inhibits mitophagy and alleviates the injury in hypoxia-induced trophoblast cells

Preeclampsia (PE) is a pregnancy disorder characterized by excessive trophoblast cell death. This study aims to explore the exact mechanism of the ubiquitination level of FUN14 domain containing 1 (FUNDC1) in mitophagy and injury in hypoxic trophoblast cells. In this study, HTR-8/SVneo trophoblast cells were cultured under normoxic and hypoxic conditions and PE mouse model was established. We found low ubiquitination level of FUNDC1 in hypoxic trophoblast cells and placenta of pregnant women with PE. Proteasome inhibitor MG-132 and protease activator MF-094 were added into HTR-8/SVneo trophoblast cells. Proteasome inhibitor MG-132 decreased FUNDC1 ubiquitination level while protease activator MF-094 increased FUNDC1 ubiquitination level. Inhibition of FUNDC1 ubiquitination promoted mitophagy and mitochondrial membrane potential (Δψm) in normoxic trophoblast cells, increased levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased levels of glutathione (GSH) and superoxide dismutase (SOD). In addition, FUNDC1 ubiquitination alleviated cell injury in PE mice in vivo. In conclusion, increased FUNDC1 ubiquitination level inhibited mitophagy and Δψm changes in hypoxic trophoblast cells, and thus alleviated oxidative injury.

Novel highly selective inhibitors of ubiquitin specific protease 30 (USP30) accelerate mitophagy

Mitophagy is one of the processes that cells use to maintain overall health. An E3 ligase, parkin, ubiquitinates mitochondrial proteins prior to their degradation by autophagasomes. USP30 is an enzyme that de-ubiquitinates mitochondrial proteins; therefore, inhibiting this enzyme could foster mitophagy. Herein, we disclose the structure-activity relationships (SAR) within a novel series of highly selective USP30 inhibitors. Two structurally similar compounds, MF-094 (a potent and selective USP30 inhibitor) and MF-095 (a significantly less potent USP30 inhibitor), serve as useful controls for biological evaluation. We show that MF-094 increases protein ubiquitination and accelerates mitophagy.