Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

The mitophagy pathway and its implications in human diseases.

Mitochondria are dynamic organelles with multiple functions. They participate in necrotic cell death and programmed apoptotic, and are crucial for cell metabolism and survival. Mitophagy serves as a cytoprotective mechanism to remove superfluous or dysfunctional mitochondria and maintain mitochondrial fine-tuning numbers to balance intracellular homeostasis. Growing evidences show that mitophagy, as an acute tissue stress response, plays an important role in maintaining the health of the mitochondrial network. Since the timely removal of abnormal mitochondria is essential for cell survival, cells have evolved a variety of mitophagy pathways to ensure that mitophagy can be activated in time under various environments. A better understanding of the mechanism of mitophagy in various diseases is crucial for the treatment of diseases and therapeutic target design. In this review, we summarize the molecular mechanisms of mitophagy-mediated mitochondrial elimination, how mitophagy maintains mitochondrial homeostasis at the system levels and organ, and what alterations in mitophagy are related to the development of diseases, including neurological, cardiovascular, pulmonary, hepatic, renal disease, etc., in recent advances. Finally, we summarize the potential clinical applications and outline the conditions for mitophagy regulators to enter clinical trials. Research advances in signaling transduction of mitophagy will have an important role in developing new therapeutic strategies for precision medicine.

Tetramethylpyrazine attenuated bupivacaine-induced neurotoxicity in SH-SY5Y cells through regulating apoptosis, autophagy and oxidative damage.

Background: Bupivacaine (BUP) acts as a local anesthetic, which is extensively used for clinical patients but could generate neurotoxicity in neurons. Tetramethylpyrazine (TET) exhibits strong neuron protective effects against neurotoxicity. Hence, we investigate the effect of TET on BUP-induced neurotoxicity in SH-SY5Y cells. Methods: CCK-8 assay was used to detect cell proliferation in SH-SY5Y cells. In addition, Western blotting was used to examine Bax, Bcl-2, active caspase 3, LC3II, Beclin 1 and p-62 protein levels in cells. Moreover, ELISA assay was used to detect the levels of total glutathione (GS), superoxide dismutase (SOD) and malondialdehyde (MDA) in cells. Results: In this study, we found that TET attenuated the neurotoxicity of BUP on SH-SY5Y cells. Meanwhile, TET alleviated BUP-induced apoptosis in SH-SY5Y cell via decreasing the expressions of active caspase-3 and Bax and increasing the expression of Bcl-2. In addition, monodansylcadaverine staining assay and Western blotting results confirmed that TET induced autophagy in SH-SY5Y cells via increasing the LC3II/I and Beclin 1 levels. Furthermore, TET attenuated BUP-induced oxidative damage in SH-SY5Y cells via upregulation of the levels of total GS and SOD and downregulation of the level of MDA. Interesting, the protective effects of TET against BUP-induced neurotoxicity in SH-SY5Y cells were reversed by autophagy inhibitor 3-methyladenine (3MA). Conclusion: These data indicated that TET may play a neuroprotective role via inhibiting apoptosis and inducing autophagy in SH-SY5Y cells. Therefore, TET may be a potential agent for the treatment of human neurotoxicity induced by BUP.

Dexmedetomidine preconditioning plays a neuroprotective role and suppresses TLR4/NF-κB pathways model of cerebral ischemia reperfusion.

BACKGROUND: Dexmedetomidine has been reported to play an efficient role on multi-organ protection. Our study aims to investigate the neuroprotective of dexmedetomidine preconditioning on cerebral ischemic reperfusion (I/R) injury and investigate the underlining signaling mechanisms. METHODS: Cerebral I/R models were established with SD rats through middle cerebral artery occlusion (MCAO). After 2h of ischemia followed by 7days of reperfusion, the degree of cerebral tissue injury was detected by HE, Nissl and TUNEL staining. Glial fibrillary acidic protein (GFAP) positive and TNF-α positive cells were stained by immunohistochemistry and counted under microscope. TLR4, NF-κB and TIR-domain containing adapter-inducing interferon-ß (TRIF) expression were detected by real time PCR and western blot. RESULTS: Dexmedetomidine preconditioning markedly prevented the ischemia-induced cellular damage observed from HE and Nissl staining in hippocampus and cortex. Dexmedetomidine observably decreased the number of apoptotic cells in TUNEL staining. Besides, yohimbine could specifically suppress the protective effect of dexmedetomidine. GFAP expression was distinctly inhibited by dexmedetomidine preconditioning (10µg/kg, 20µg/kg) in cerebral ischemia area. Dexmedetomidine preconditioning inhibited the expression of TLR4 and NF-κB and increased that of TRIF. CONCLUSION: The results of this study suggest that dexmedetomidine preconditioning plays a neuroprotective role against I/R injury. Dexmedetomidine might suppress TLR4/NF-??B pathway and drive TLR4/TRIF signaling pathway to reduce the inflammatory injury.

Enantioselective synthesis of axially chiral multifunctionalized biaryls via asymmetric Suzuki-Miyaura coupling.

Substituted 2-formylarylboronic acids were successfully employed as substrates for asymmetric Suzuki-Miyaura coupling. By virtue of the coupling with dialkoxyphosphinyl substituted naphthyl bromides and 2-nitronaphthalen-1-yl triflouromethanesulfonate, a series of novel multifunctionalized axially chiral biaryls were prepared in 53-97% yields with up to 97% ee using palladium-Cy-MOP as the catalyst. The methodology provides a highly efficient and practical strategy for the synthesis of novel multifunctionalized axially chiral biaryls.

Highly efficient synthesis of a class of novel chiral-bridged atropisomeric monophosphine ligands via simple desymmetrization and their applications in asymmetric Suzuki-Miyaura coupling reaction.

A series of novel chiral-bridged atropisomeric monophosphine ligands were synthesized via convenient and simple pathways. The prepared ligands, especially for ligand 7d, were found to be highly effective in the Pd-catalyzed Suzuki-Miyaura coupling reaction. The steric hindrance and electronic effect of substrates on the reactivity and enantioselectivity were explored preliminarily.

Additional new tetracyclic tetraterpenoid: methyl tortuoate D from soft coral Sarcophyton tortuosum.

Under the guidance of chemical prescreening by direct infusion electrospray ionization mass spectrometry (DI ESI-MS), a new tetracyclic tetraterpenoid, methyl tortuoate D (1) was isolated from the soft coral Sarcophyton tortuosum collected from the South China Sea. Its structure and relative stereochemistry were established by MS, and 1D- and 2D-NMR spectroscopic techniques.