Potential roles of UCH family deubiquitinases in tumorigenesis and chemical inhibitors developed against them.

Deubiquitinating enzymes (DUBs) are a large group of proteases that reverse ubiquitination process and maintain protein homeostasis. The DUBs have been classified into seven subfamilies according to their primary sequence and structural similarity. As a small subfamily of DUBs, the ubiquitin C-terminal hydrolases (UCHs) subfamily only contains four members including UCHL1, UCHL3, UCHL5, and BRCA1-associated protein-1 (BAP1). Despite sharing the deubiquitinase activity with a similar catalysis mechanism, the UCHs exhibit distinctive biological functions which are mainly determined by their specific subcellular localization and partner substrates. Besides, growing evidence indicates that the UCH enzymes are involved in human malignancies. In this review, the structural information and biological functions of the UCHs are briefly described. Meanwhile, the roles of these enzymes in tumorigenesis and the discovered inhibitors against them are also summarized to give an insight into the cancer therapy with the potential alternative strategy.

PPIA dictates NRF2 stability to promote lung cancer progression.

Nuclear factor erythroid 2-related factor 2 (NRF2) hyperactivation has been established as an oncogenic driver in a variety of human cancers, including non-small cell lung cancer (NSCLC). However, despite massive efforts, no specific therapy is currently available to target NRF2 hyperactivation. Here, we identify peptidylprolyl isomerase A (PPIA) is required for NRF2 protein stability. Ablation of PPIA promotes NRF2 protein degradation and blocks NRF2-driven growth in NSCLC cells. Mechanistically, PPIA physically binds to NRF2 and blocks the access of ubiquitin/Kelch Like ECH Associated Protein 1 (KEAP1) to NRF2, thus preventing ubiquitin-mediated degradation. Our X-ray co-crystal structure reveals that PPIA directly interacts with a NRF2 interdomain linker via a trans-proline 174-harboring hydrophobic sequence. We further demonstrate that an FDA-approved drug, cyclosporin A (CsA), impairs the interaction of NRF2 with PPIA, inducing NRF2 ubiquitination and degradation. Interestingly, CsA interrupts glutamine metabolism mediated by the NRF2/KLF5/SLC1A5 pathway, consequently suppressing the growth of NRF2-hyperactivated NSCLC cells. CsA and a glutaminase inhibitor combination therapy significantly retard tumor progression in NSCLC patient-derived xenograft (PDX) models with NRF2 hyperactivation. Our study demonstrates that targeting NRF2 protein stability is an actionable therapeutic approach to treat NRF2-hyperactivated NSCLC.

Addition of α-synuclein aggregates to the intestinal environment recapitulates Parkinsonian symptoms in model systems.

The gut-brain axis plays a vital role in Parkinson's disease (PD). The mechanisms of gut-brain transmission mainly focus on α-synuclein deposition, intestinal inflammation and microbiota function. A few studies have shown the trigger of PD pathology in the gut. α-Synuclein is highly conserved in food products, which was able to form ß-folded aggregates and to infect the intestinal mucosa. In this study we investigated whether α-synuclein-preformed fibril (PFF) exposure could modulate the intestinal environment and induce rodent models replicating PD pathology. We first showed that PFF could be internalized into co-cultured Caco-2/HT29/Raji b cells in vitro. Furthermore, we demonstrated that PFF perfusion caused the intestinal inflammation and activation of enteric glial cells in an ex vivo intestinal organ culture and in an in vivo intestinal mouse coloclysis model. Moreover, we found that PFF exposure through regular coloclysis induced PD pathology in wild-type (WT) and A53T α-synuclein transgenic mice with various phenotypes. Particularly in A53T mice, PFF induced significant behavioral disorders, intestinal inflammation, α-synuclein deposition, microbiota dysbiosis, glial activation as well as degeneration of dopaminergic neurons in the substantia nigra. In WT mice, however, the PFF induced only mild behavioral abnormalities, intestinal inflammation, α-synuclein deposition, and glial activation, without significant changes in microbiota and dopaminergic neurons. Our results reveal the possibility of α-synuclein aggregates binding to the intestinal mucosa and modeling PD in mice. This study may shed light on the investigation and early intervention of the gut-origin hypothesis in neurodegenerative diseases.

Screening and optimization of phage display cyclic peptides against the WDR5 WBM site.

Of the various WD40 family proteins, WDR5 is a particularly important multifunctional adaptor protein that can bind to several protein complexes to regulate gene activation, so it was considered as a promising epigenetic target in anti-cancer drug development. Despite many inhibitors having been discovered directing against the arginine-binding cavity in WDR5 called the WIN site, the side hydrophobic cavity called the WBM site receives rather scant attention. Herein, we aim to obtain novel WBM-targeted peptidic inhibitors with high potency and selectivity. We employed two improved biopanning approaches with a disulfide-constrained cyclic peptide phage library containing 7 randomized residues and identified several peptides with micromole binding activity by docking and binding assay. To further optimize the stability and activity, 9 thiol-reactive chemical linkers were utilized in the cyclization of the candidate peptide DH226027, which had good binding affinity. This study provides an effective method to discover potent peptides targeting protein-protein interactions and highlights a broader perspective of peptide-mimic drugs.

A hydrogen-deuterium exchange mass spectrometry-based protocol for protein-small molecule interaction analysis.

Protein-small molecule interaction is vital in regulating protein functions and controlling various cellular processes. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein-small molecule interactions, however, to accurately probe the conformational dynamics of the protein upon small molecule binding, the HDX-MS experimental conditions should be carefully controlled and optimized. Here, we present the detailed continuous-labeling, bottom-up HDX-MS protocol for studying protein-small molecule interactions. We took a side-by-side HDX kinetics comparison of the Hsp90N protein with or without the treatment of small molecules (i.e., Radicicol, Geldanamycin) for displaying conformational changes induced by molecular interactions between Hsp90N and small molecules. Our sensitive and robust experimental protocol can facilitate the novice to quickly carry out the structural characterization of protein-small molecule interactions.

Otilonium Bromide acts as a selective USP28 inhibitor and exhibits cytotoxic activity against multiple human cancer cell lines.

USP28 contributes to tumorigenesis through modulating the lifespan of oncogenic factors such as c-Myc and ΔNp63, and it has been identified as a potential target for anti-cancer drug development. Currently, although quite a number of USP28 inhibitors have been developed, they all are still in preclinical research stage. Besides, none of them exhibits satisfying inhibition selectivity against USP28 over its closest homologue USP25. Here in this manuscript, a high-throughput screening aiming to discover USP28 inhibitors with novel scaffold and enhanced inhibition selectivity were conducted. After the primary screening and the second round of validation, Otilonium Bromide, an approved drug for treating irritable bowel syndrome, was identified to inhibit USP28's activity with the IC50 value at 6.90 ± 0.90 µM. Besides, the drug exhibits a 3-4 folds inhibition selectivity against USP28 over USP25. According to the enzymatic kinetics analysis data and the hydrogen-deuterium exchange mass spectrometry results, Otilonium Bromide could bind to the allosteric pocket of USP28 and inhibit its activity in a reversible and non-competitive mode. The following performed cell-based assays revealed that the drug could cause cytotoxicity against human colorectal cancer cells and lung squamous carcinoma cells potentially through down-regulating USP28's oncogenic substrates c-Myc and/or ΔNp63. Meanwhile, since Otilonium Bromide has been found to preferentially distribute to gastrointestinal tissues, we then evaluated its potential in the combination treatment of colorectal cancer cells with Regorafenib, which is an approved drug for colorectal cancer therapy. As expected, Otilonium Bromide could significantly enhance the sensitivity of colorectal cancer cells to Regorafenib.

Human Aha1's N-terminal extension confers it holdase activity in vitro.

Molecular chaperones are key components of protein quality control system, which plays an essential role in controlling protein homeostasis. Aha1 has been identified as a co-chaperone of Hsp90 known to strongly accelerate Hsp90's ATPase activity. Meanwhile, it is reported that Aha1 could also act as an autonomous chaperone and protect stressed or disordered proteins from aggregation. Here, in this article, a series of in vitro experiments were conducted to verify whether Aha1 has a non-Hsp90-dependent holdase activity and to elucidate the associated molecular mechanism for substrate recognition. According to the results of the refolding assay, the highly conserved N-terminal extension spanning M1 to R16 in Aha1 from higher eukaryotes is responsible for the holdase activity of the protein. As revealed by the NMR data, Aha1's N-terminal extension mainly adopts a disordered conformation in solution and shows no tight contacts with the core structure of Aha1's N-terminal domain. Based on the intrinsically disordered structure feature and the primary sequence of Aha1's N-terminal extension, the fuzzy-type protein-protein interactions involving this specific region and the unfolded substrate proteins are expected. The following mutation analysis data demonstrated that the Van der Waals contacts potentially involving two tryptophans including W4 and W11 do not play a dominant role in the interaction between Aha1 and unfolded maltose binding protein (MBP). Meanwhile, since the high concentration of NaCl could abolish the holdase activity of Aha1, the electrostatic interactions mediated by those charged residues in Aha1's N-terminal extension are thus indicated to play a crucial role in the substrate recognition.

Pharmacological targeting of Axin2 suppresses cell growth and metastasis in colorectal cancer.

BACKGROUND AND PURPOSE: The scaffold molecule Axin2 is constitutively activated in colorectal cancer (CRC) and functions as a potent promoter of CRC behaviour. Pharmacological targeting of Axin2 may therefore exert a therapeutic effect in patients with CRC. Here, we discovered a potent small-molecule inhibitor of Axin2, based on the mechanism by which Axin2 is regulated post-translationally, and investigated its antitumour effects. EXPERIMENTAL APPROACH: Compound discovery and its inhibitory action on Axin2 protein were revealed by microscale thermophoresis, in vitro kinase assay, quantitative kinetic assay, immunoblotting/immunoprecipitation, RT-qPCR and cycloheximide pulse-chase assay. Compound antitumour effects and the underlying mechanisms were evaluated in multiple cell-based assays and mouse models. KEY RESULTS: We discovered that glycogen synthase kinase 3ß (GSK3ß) phosphorylates Axin2 at two consensus motifs and coupled Axin2 phosphorylation to its ubiquitination (mediated by the E3 ligase ß-Trcp2) and proteasomal degradation. The binding of Axin2 to GSK3ß in CRC cells is faint, which enables most of the Axin2 protein to maintain an unphosphorylated status and thereby permits the cells to preserve high levels of Axin2. Importantly, we identified a small-molecule compound CW85319 that enhances Axin2's interaction with GSK3ß via forming a high affinity for Axin2. Treatment of CRC cells with CW85319 enhanced Axin2 binding with GSK3ß, thereby promoting Axin2 phosphorylation, subsequent ubiquitination, and degradation. Furthermore, we demonstrated that CW85319 efficiently suppressed Axin2-driven CRC growth and metastasis, without eliciting side toxicity. CONCLUSIONS AND IMPLICATIONS: These findings suggest that pharmacological targeting of Axin2 by CW85319 may provide therapeutic benefits against certain human cancers, especially CRC.

Spermine is a natural suppressor of AR signaling in castration-resistant prostate cancer.

In castration-resistant prostate cancer (CRPC), clinical response to androgen receptor (AR) antagonists is limited mainly due to AR-variants expression and restored AR signaling. The metabolite spermine is most abundant in prostate and it decreases as prostate cancer progresses, but its functions remain poorly understood. Here, we show spermine inhibits full-length androgen receptor (AR-FL) and androgen receptor splice variant 7 (AR-V7) signaling and suppresses CRPC cell proliferation by directly binding and inhibiting protein arginine methyltransferase PRMT1. Spermine reduces H4R3me2a modification at the AR locus and suppresses AR binding as well as H3K27ac modification levels at AR target genes. Spermine supplementation restrains CRPC growth in vivo. PRMT1 inhibition also suppresses AR-FL and AR-V7 signaling and reduces CRPC growth. Collectively, we demonstrate spermine as an anticancer metabolite by inhibiting PRMT1 to transcriptionally inhibit AR-FL and AR-V7 signaling in CRPC, and we indicate spermine and PRMT1 inhibition as powerful strategies overcoming limitations of current AR-based therapies in CRPC.

Sequence-based drug design as a concept in computational drug design.

Drug development based on target proteins has been a successful approach in recent decades. However, the conventional structure-based drug design (SBDD) pipeline is a complex, human-engineered process with multiple independently optimized steps. Here, we propose a sequence-to-drug concept for computational drug design based on protein sequence information by end-to-end differentiable learning. We validate this concept in three stages. First, we design TransformerCPI2.0 as a core tool for the concept, which demonstrates generalization ability across proteins and compounds. Second, we interpret the binding knowledge that TransformerCPI2.0 learned. Finally, we use TransformerCPI2.0 to discover new hits for challenging drug targets, and identify new target for an existing drug based on an inverse application of the concept. Overall, this proof-of-concept study shows that the sequence-to-drug concept adds a perspective on drug design. It can serve as an alternative method to SBDD, particularly for proteins that do not yet have high-quality 3D structures available.

An ATG4B inhibitor blocks autophagy and sensitizes Sorafenib inhibition activities in HCC tumor cells.

Autophagy related 4B (ATG4B) which regulates autophagy by promoting the formation of autophagosome through reversible modification of LC3, is closely related to cancer cell growth and drug resistance, and therefore is an attractive therapeutic target. Recently, ATG4B inhibitors have been reported, yet with drawbacks including weak potency. To discover more promising ATG4B inhibitors, we developed a high-throughput screening (HTS) assay and identified a new ATG4B inhibitor named DC-ATG4in. DC-ATG4in directly binds to ATG4B and inhibits its enzyme activity with an IC50 of 3.08 ± 0.47 µM. We further confirmed that DC-ATG4in is an autophagy inhibitor and blocks autophagy induced by Sorafenib in Hepatocellular Carcinoma (HCC) cells. More importantly, combination of DC-ATG4in with Sorafenib synergized the cancer cell killing effect and proliferation inhibition activities on HCC cells. Our data suggested that inactivation of autophagy via ATG4B inhibition may be a viable strategy to sensitize existing targeted therapy such as Sorafenib in the future.

Structure-based discovery of potent USP28 inhibitors derived from Vismodegib.

Ubiquitin-specific proteases (USPs) 28 is overexpressed in multiple types of cancers. The development of potent USP28 inhibitors is still in primitive stage. We previously reported our discovery of Vismodegib as a USP28 inhibitor by screening a commercially available drug library. Herein, we report our efforts to solve the cocrystal structure of Vismodegib bound to USP28 for the first time and subsequent structure-based optimization leading to a series of Vismodegib derivatives as potent USP28 inhibitors. Based on the cocrystal structure, elaborative SARs exploration was carried out to afford much more potent USP28 inhibitors than Vismodegib. The representative compounds 9l, 9o and 9p bearing high potency on USP28 showed high selectivity over USP2, USP7, USP8, USP9x, UCHL3 and UCHL5. The detailed cellular assay suggested that compounds 9l, 9o and 9p could cause cytotoxicity in both human colorectal cancer and lung squamous carcinoma cells and significantly enhance the sensitivity of colorectal cancer cells to Regorafenib. Further immunoblotting analysis indicated that compounds 9l, 9o and 9p could dose-dependently down-regulate the cellular level of c-Myc through ubiquitin-proteasome system and anti-cancer effects could mainly be attributed to their inhibition on USP28 but not involving the Hedgehog-Smoothened pathway. Thus, our work provided a series of novel and potent USP28 inhibitors derived from Vismodegib and may contribute to the development of USP28 inhibitors.

Performance comparison of three scaling algorithms in NMR-based metabolomics analysis.

Unit variance (UV) scaling, mean centering (CTR) scaling, and Pareto (Par) scaling are three commonly used algorithms in the preprocessing of metabolomics data. Based on our NMR-based metabolomics studies, we found that the clustering identification performances of these three scaling methods were dramatically different as tested by the spectra data of 48 young athletes' urine samples, spleen tissue (from mice), serum (from mice), and cell (from Staphylococcus aureus) samples. Our data suggested that for the extraction of clustering information, UV scaling could serve as a robust approach for NMR metabolomics data for the identification of clustering analysis even with the existence of technical errors. However, for the purpose of discriminative metabolite identification, UV scaling, CTR scaling, and Par scaling could equally extract discriminative metabolites efficiently based on the coefficient values. Based on the data presented in this study, we propose an optimal working pipeline for the selection of scaling algorithms in NMR-based metabolomics analysis, which has the potential to serve as guidance for junior researchers working in the NMR-based metabolomics research field.

Discovery of new small molecule inhibitors of the BPTF bromodomain.

Chromatin remodeling regulates many basic cellular processes, such as gene transcription, DNA repair, and programmed cell death. As the largest member of nucleosome remodeling factor (NURF), BPTF plays a vital role in the occurrence and development of cancer. Currently, BPTF bromodomain inhibitors are still in development. In this study, by conducting homogenous time-resolved fluorescence resonance energy transfer (HTRF) assay, we identified a potential, novel BPTF inhibitor scaffold Sanguinarine chloride with the IC50 value of 344.2 ± 25.1 nM. Biochemical analysis revealed that compound Sanguinarine chloride exhibited high binding affinity to the BPTF bromodomain. Molecular docking predicted the binding mode of Sanguinarine chloride and elucidated the activities of its derivatives. Moreover, Sanguinarine chloride showed a potent anti-proliferative effect in MIAPaCa-2 cells and inhibited the expression of BPTF target gene c-Myc. Taken together, Sanguinarine chloride provides a qualified chemical tool for developing potent BPTF bromodomain inhibitors.

Oxidative stress, dysfunctional energy metabolism, and destabilizing neurotransmitters altered the cerebral metabolic profile in a rat model of simulated heliox saturation diving to 4.0 MPa.

The main objective of the present study was to determine metabolic profile changes in the brains of rats after simulated heliox saturated diving (HSD) to 400 meters of sea water compared to the blank controls. Alterations in the polar metabolome in the rat brain due to HSD were investigated in cortex, hippocampus, and striatum tissue samples by applying an NMR-based metabolomic approach coupled with biochemical detection in the cortex. The reduction in glutathione and taurine levels may hypothetically boost antioxidant defenses during saturation diving, which was also proven by the increased malondialdehyde level, the decreased superoxide dismutase, and the decreased glutathione peroxidase in the cortex. The concomitant decrease in aerobic metabolic pathways and anaerobic metabolic pathways comprised downregulated energy metabolism, which was also proven by the biochemical quantification of the metabolic enzymes Na-K ATPase and LDH in cerebral cortex tissue. The significant metabolic abnormalities of amino acid neurotransmitters, such as GABA, glycine, and aspartate, decreased aromatic amino acids, including tyrosine and phenylalanine, both of which are involved in the metabolism of dopamine and noradrenaline, which are downregulated in the cortex. Particularly, a decline in the level of N-acetyl aspartate is associated with neuronal damage. In summary, hyperbaric decompression of a 400 msw HSD affected the brain metabolome in a rat model, potentially including a broad range of disturbing amino acid homeostasis, metabolites related to oxidative stress and energy metabolism, and destabilizing neurotransmitter components. These disturbances may contribute to the neurochemical and neurological phenotypes of HSD.

Sodium Oligomannate Electrostatically Binds to Aß and Blocks Its Aggregation.

GV-971 (sodium oligomannate) is a China Food and Drug Administration (CFDA)-approved drug for treating Alzheimer's disease, and it could inhibit Aß fibril formation in vitro and in mouse studies. To elucidate the mechanisms for understanding how GV-971 modulates Aß's aggregation, we conducted a systematic biochemical and biophysical study of Aß40/Aß42:GV-971 systems. The integrating analysis of previously published data and our results suggests that the multisite electrostatic interactions between GV-971's carboxylic groups and Aß40/Aß42's three histidine residues might play a dominant role in driving the binding of GV-971 to Aß. The fuzzy-type electrostatic interactions between GV-971 and Aß are expected to protect Aß from aggregation potentially through breaking the histidine-mediated inter-Aß electrostatic interactions. Meanwhile, since GV-971's binding exhibited a slight downregulation effect on the flexibility of Aß's histidine-colonized fragment, which potentially favors Aß aggregation, we conclude that the dynamics alteration plays a minor role in GV-971's modulation on Aß aggregation.

Pharmacological characterization of the small molecule 03A10 as an inhibitor of α-synuclein aggregation for Parkinson's disease treatment.

Aggregation of α-synuclein, a component of Lewy bodies (LBs) or Lewy neurites in Parkinson's disease (PD), is strongly linked with disease development, making it an attractive therapeutic target. Inhibiting aggregation can slow or prevent the neurodegenerative process. However, the bottleneck towards achieving this goal is the lack of such inhibitors. In the current study, we established a high-throughput screening platform to identify candidate compounds for preventing the aggregation of α-synuclein among the natural products in our in-house compound library. We found that a small molecule, 03A10, i.e., (+)-desdimethylpinoresinol, which is present in the fruits of Vernicia fordii (Euphorbiaceae), modulated aggregated α-synuclein, but not monomeric α-synuclein, to prevent further elongation of α-synuclein fibrils. In α-synuclein-overexpressing cell lines, 03A10 (10 µM) efficiently prevented α-synuclein aggregation and markedly ameliorated the cellular toxicity of α-synuclein fibril seeds. In the MPTP/probenecid (MPTP/p) mouse model, oral administration of 03A10 (0.3 mg· kg-1 ·d-1, 1 mg ·kg-1 ·d-1, for 35 days) significantly alleviated behavioral deficits, tyrosine hydroxylase (TH) neuron degeneration and p-α-synuclein aggregation in the substantia nigra (SN). As the Braak hypothesis postulates that the prevailing site of early PD pathology is the gastrointestinal tract, we inoculated α-synuclein preformed fibrils (PFFs) into the mouse colon. We demonstrated that α-synuclein PFF inoculation promoted α-synuclein pathology and neuroinflammation in the gut and brain; oral administration of 03A10 (5 mg· kg-1 ·d-1, for 4 months) significantly attenuated olfactory deficits, α-synuclein accumulation and neuroinflammation in the olfactory bulb and SN. We conclude that 03A10 might be a promising drug candidate for the treatment of PD. 03A10 might be a novel drug candidate for PD treatment, as it inhibits α-synuclein aggregation by modulating aggregated α-synuclein rather than monomeric α-synuclein to prevent further elongation of α-synuclein fibrils and prevent α-synuclein toxicity in vitro, in an MPTP/p mouse model, and PFF-inoculated mice.

Dipyridamole interacts with the N-terminal domain of HSP90 and antagonizes the function of the chaperone in multiple cancer cell lines.

Molecular chaperone HSP90 has been considered as a promising target for anti-cancer drug development for years. However, due to the heat shock response induced by the ATP competitive inhibitors against HSP90, the therapeutic efficacies of the compounds are compromised, which consequently restricts the clinical use of HSP90-targeted inhibitors. Therefore, there is a need to discover novel HSP90-targeted modulators which exhibit acceptable inhibition activity against the chaperone and do not induce significant heat shock response in the meantime. Here in this study, we firstly developed a tip-based affinity selection-mass spectrometry platform with optimized experimental conditions/parameters for HSP90-targeted active compound screening, and then applied it to fish out inhibitors against HSP90 from a collection of 2,395 compounds composed of FDA-approved drugs and drug candidates. Dipyridamole, which acts as an anti-thrombotic agent by modulating multiple targets and has a long history of safe use, was identified to interact with HSP90's N-terminal domain. The following conducted biophysical and biochemical experiments demonstrated that Dipyridamole could bind to HSP90's ATP binding pocket and function as an ATP competitive inhibitor of the chaperone. Finally, cellular-based assays including CESTA, cell viability assessment and proteomic analysis etc. were performed to evaluate whether the interaction between HSP90 and Dipyridamole contributes to the anti-tumor effects of the compound. We then found that Dipyridamole inhibits the growth and proliferation of human cancer cells by downregulating cell cycle regulators and upregulating apoptotic cell signaling, which are potentially mediated by the binding of Dipyridamole to HSP90 and to PDEs (phosphodiesterases), respectively.

(-)-Epigallocatechin Gallate is a Noncompetitive Inhibitor of NAD Kinase.

Nicotinamide adenine dinucleotide kinase (NADK) controls the intracellular NADPH content and provides reducing power for the synthesis of macromolecules and anti-ROS. Moreover, NADK is considered to be a synthetic lethal gene for KRAS mutations. To discover NADK-targeted probes, a high-throughput screening assay was established and optimized with a Z factor of 0.71. The natural product (-)-epigallocatechin gallate (EGCG) was found to be a noncompetitive inhibitor of NADK with K i = 3.28 ± 0.32 µΜ. The direct binding of EGCG to NADK was determined by several biophysical methods, including NMR spectroscopy, surface plasmon resonance (SPR) assay, and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The SPR assay showed a K d of 1.78 ± 1.15 µΜ. The HDX-MS experiment showed that EGCG was bound at the non-substrate-binding sites of NADK. Besides, binding mode prediction and derivative activity analysis revealed a potential structure-activity relationship between EGCG and NADK. Furthermore, EGCG can specifically inhibit the proliferation of KRAS-mutated lung cancer cell lines without affecting KRAS wild-type lung cancer cell lines.

7-aminocephalosporanic acid, a novel HSP90ß inhibitor, attenuates HFD-induced hepatic steatosis.

Hepatic steatosis is one of the most important causes of liver disease worldwide. Heat shock protein 90 (HSP90) is essential for numerous client proteins. Recently, more attention was focused on increased HSP90 levels in hepatic steatosis, especially HSP90ß. Thus, great efforts have been made to develop HSP90ß inhibitors, and most natural inhibitors are derived from microorganisms. In this study, using microarray chips and surface pasmon resonance (SPR) technology, we screened 189 antibiotics in order to obtain an inhibitor directly binding to the non-N-terminal domain of HSP90ß. Finally, we discovered an antibiotic, 7-aminocephalosporanic acid (7ACA), with a KD value of 6.201 µM between 7ACA and non-N-terminal domain of HSP90ß. Besides, 7ACA was predicted to interact with the middle domain (MD) of HSP90ß. In HepG2 cells, we found that 7ACA reduced cellular total cholesterol (TC) and triglyceride (TG) by decreasing sterol regulatory element-binding proteins (SREBPs). In HFD fed mice, administration of 7ACA (5, 10, and 25 mg kg-1 d-1, ig, for 12 weeks) dose-dependently decreased serum TC and TG and played an important role in protecting liver and adipose tissue from lipid accumulation. In conclusion, our study demonstrated that antibiotic 7ACA, as an HSP90ß middle domain inhibitor, was promising for the development of lipid-lowering drugs.