Dual inhibitors of DNMT and HDAC remodels the immune microenvironment of colorectal cancer and enhances the efficacy of anti-PD-L1 therapy.

Colorectal cancer is the second most prevalent and deadly cancer worldwide. The emergence of immune checkpoint therapy has provided a revolutionary strategy for the treatment of solid tumors. However, less than 5% of colorectal cancer patients respond to immune checkpoint therapy. Thus, it is of great scientific significance to develop "potentiators" for immune checkpoint therapy. In this study, we found that knocking down different DNMT and HDAC isoforms could increase the expression of IFNs in colorectal cancer cells, which can enhance the effectiveness of immune checkpoint therapy. Therefore, the combined inhibition of DNMT and HDAC cloud synergistically enhance the effect of immunotherapy. We found that dual DNMT and HDAC inhibitors C02S could inhibit tumor growth in immunocompetent mice but not in immunocompromised nude mice, which indicates that C02S exerts its antitumor effects through the immune system. Mechanistically, C02S could increase the expression of ERVs, which generated the intracellular levels of dsRNA in tumor cells, and then promotes the expression of IFNs through the RIG-I/MDA5-MAVS signaling pathway. Moreover, C02S increased the immune infiltration of DCs and T cells in microenvironment, and enhanced the efficacy of anti-PD-L1 therapy in MC38 and CT26 mice model. These results confirmed that C02S can activate IFNs through the RIG-I/MDA5-MAVS signaling pathway, remodel the tumor immune microenvironment and enhance the efficacy of immune checkpoint therapy, which provides new evidence and solutions for the development of "potentiator" for colorectal cancer immunotherapy.

Rapid exosome isolation and in situ multiplexed detection of exosomal surface proteins and microRNAs on microfluidic platform.

Exosomes are considered as promising biomarkers for early cancer diagnosis and prognosis. However, the majority of the research studies focused on a single type of exosomal biomarkers, which cannot comprehensively reflect the state of cancer for accurate diagnosis. To address this problem, we presented a ship-shaped microfluidic device containing a microcolumn array for simultaneous in situ detection of exosomal surface proteins and miRNAs. Exosomes were first captured in the microchannels modified with CD63 protein aptamer. Exosomal surface proteins and miRNAs were simultaneously detected in four parallel channels to avoid the interference of fluorescent signals using specific aptamers labeled by Cy5 and catalytic hairpin assembly (CHA) based signal amplification strategy. The limit of detection for multiplexed markers in exosomes was 83 exosomes per µL, which is comparable to previously reported methods. Through quantitative analysis of two disease-specific surface proteins and miRNAs derived from different cancer cells and clinical serum samples, different cancer subtypes as well as cancer patients and healthy people could be significantly distinguished. These results suggest that this simple, highly sensitive, and more accurate analytical strategy by simultaneous in situ profiling of different types of exosomal biomarkers has potential applications in cancer diagnosis and stage monitoring.

Design, synthesis and biological evaluation of pyrimidine base hydroxamic acid derivatives as dual JMJD3 and HDAC inhibitors.

The Jumonji domain-containing protein demethylase 3 (JMJD3) and histone deacetylase (HADC) are related to various cancers and regard as antitumor targets for drug discovery. In this study, based on rational drug design strategy, we designed and synthesized a series of pyrimidine derivatives with hydroxamic acid as novel dual JMJD3 and HDAC inhibitors for synergistic cancer treatment. Compound A5b exhibited inhibitory potency against JMJD3 and HDAC1/6 simultaneously and favorable cytotoxicity against human cancer cells such as A549 and U937. Furthermore, mechanistic studies showed that A5b treatment in A549 cells increased the hypermethylation of histone H3K27 and hyperacetylation of H3K9, suppressed clonogenicity, migration and invasion of cancer cells. Besides, A5b induced apoptosis via the cleavage of caspase-7 and PARP, and G1 cell cycle arrest via upregulated p21 expression. All these results suggested that A5b was the first dual inhibitor against JMJD3 and HDAC and can be a potential compound for cancer therapy.

Generic Plug-and-Play Strategy for High-Throughput Analysis of PTM-Mediated Protein Complexes.

Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.

Quinazoline-based hydroxamic acid derivatives as dual histone methylation and deacetylation inhibitors for potential anticancer agents.

Cancer is a common malignant disease with complex signaling networks, which means it is unmanageable to cancer therapy by using single classical targeted drug. Recently, dual- or multitarget drugs have emerged as a promising option for cancer therapies. Although many multifunctional compounds targeting HDAC have been validated, as far as we know, there is no molecule targeting GLP and HDAC synchronously. In the present work, we designed and synthesized a series of quinazoline-based hydroxamic acid derivatives as dual GLP and HDAC inhibitors. These hybrid compounds showed potent enzymatic inhibitory activities against GLP and HDAC1/6 with IC50 values in the nanomolar range of less than 190 nM. Furthermore, most of our compounds displayed significant broad spectrum cytotoxic activities apart from D3 and D8 against all the tested cancer cells with IC50 values less than 50 µM. D1, D6 and D7 showed more potent cytotoxic activities than D2, D4 and D5 in those cancer cells. Especially, compound D7 showed potent inhibitory potency activity against both GLP and HDAC1/6 with IC50 values of 1.3, 89, 13 nM. Besides, D7 exhibited the most potent antiproliferative activity against all the tested cancer cells. Further evaluations indicated that D7 could inhibit the methylation and deacetylation of H3K9 on protein level. Moreover, D7 could induce cancer cell apoptosis, G0/G1 cell cycle arrest, and partly block migration and invasion. All these thorough evaluations warranted D7 as a promising lead compound worth further optimization and development for cancer therapy.

Photoaffinity-engineered protein scaffold for systematically exploring native phosphotyrosine signaling complexes in tumor samples.

Phosphotyrosine (pTyr)-regulated protein complexes play critical roles in cancer signaling. The systematic characterization of these protein complexes in tumor samples remains a challenge due to their limited access and the transient nature of pTyr-mediated interactions. We developed a hybrid chemical proteomics approach, termed Photo-pTyr-scaffold, by engineering Src homology 2 (SH2) domains, which specifically bind pTyr proteins, with both trifunctional chemical probes and genetic mutations to overcome these challenges. Dynamic SH2 domain-scaffolding protein complexes were efficiently cross-linked under mild UV light, captured by biotin tag, and identified by mass spectrometry. This approach was successfully used to profile native pTyr protein complexes from breast cancer tissue samples on a proteome scale with high selectivity, achieving about 100 times higher sensitivity for detecting pTyr signaling proteins than that afforded by traditional immunohistochemical methods. Among more than 1,000 identified pTyr proteins, receptor tyrosine kinase PDGFRB expressed on cancer-associated fibroblasts was validated as an important intercellular signaling regulator with poor expression correlation to ERBB2, and blockade of PDGFRB signaling could efficiently suppress tumor growth. The Photo-pTyr-scaffold approach may become a generic tool for readily profiling dynamic pTyr signaling complexes in clinically relevant samples.

High-Throughput and Integrated Chemical Proteomic Approach for Profiling Phosphotyrosine Signaling Complexes.

Phosphotyrosine (pTyr) signaling complexes are important resources of biomarkers and drug targets which often need to be profiled with enough throughput. Current profiling approaches are not feasible to meet this need due to either biased profiling by antibody-based detection or low throughput by traditional affinity purification-mass spectrometry approach (AP-MS), as exemplified by our previously developed photo-pTyr-scaffold approach. To address these limitations, we developed a 96-well microplate-based sample preparation and fast data independent proteomic analysis workflow. By assembling the photo-pTyr-scaffold probe into a 96-well microplate, we achieved steric hindrance-free photoaffinity capture of pTyr signaling complexes, selective enrichment under denaturing conditions, and efficient in-well digestion in a fully integrated manner. EGFR signaling complex proteins could be efficiently captured and identified by using 300 times less cell lysate and 100 times less photo-pTyr-scaffold probe as compared with our previous approach operated in an Eppendorf tube. Furthermore, the lifetime of the photo-pTyr-scaffold probe in a 96-well microplate was significantly extended from 1 week up to 1 month. More importantly, by combining with high-flow nano LC separation and data independent acquisition on the Q Exactive HF-X mass spectrometer, LC-MS time could be significantly reduced to only 35 min per sample without increasing sample loading amount and compromising identification and quantification performance. This new high-throughput proteomic approach allowed us to rapidly and reproducibly profile dynamic pTyr signaling complexes with EGF stimulation at five time points and EGFR inhibitor treatment at five different concentrations. We are therefore optimized for its generic application in biomarkers discovery and drug screening in a high-throughput fashion.

High-Throughput Phosphotyrosine Protein Complexes Screening by Photoaffinity-Engineered Protein Scaffold-Based Forward-Phase Protein Array.

Low-abundance phosphotyrosine (pTyr)-mediated signaling protein complexes play critical roles in cancer signaling. The precise and comprehensive profiling of these pTyr-mediated protein complexes remains challenging because of their dynamic nature and weak binding affinity. Taking advantage of the SH2 domains modified with trifunctional chemical probes and genetic mutations (termed Photo-pTyr-scaffold), we developed a Photo-pTyr-scaffold-based forward-phase protein array that can be used to specifically capture complexes by developing an engineered SH2 domain, photoaffinity cross-linking, and antibody-based measuring weak pTyr-mediated protein complexes from complex biological samples in a 96-well microplate format. This platform demonstrated good precision for quantitation (R2 = 0.99) and high sensitivity by which only 5 µg of whole cell lysates is needed. We successfully applied the technology for profiling the dynamic EGF-stimulation-dependent EGFR signaling protein complexes across four different time courses (i.e., 0, 2, 5, 10, and 30 min) in a high-throughput manner. We further evaluated the modulation of EGFR-GRB2-SHC1 protein complexes by FDA-approved EGFR kinase inhibitor erlotinib, demonstrating the feasibility of this approach for high-throughput drug screening. The Photo-pTyr-scaffold-based forward-phase protein array could be generically applicable for exploring the dynamic pTyr signaling complexes in various biological systems and screening for related drugs in a high-throughput manner.

Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.

Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmembrane proteins such as EGFR and high quantification precision for profiling temporal interaction network of key EGFR signaling protein GRB2 across four time points of EGF treatment. More importantly, the integration feature allows minimum sample lose and helps the development of an ideal AP-MS workflow for studying endogenous protein complexes by the CRISPR Cas9 technology for the first time. By generating endogenously expressed bait protein fused with affinity tag, protein complexes associated with endogenous Integrin-linked kinase (ILK) was identified with much higher selectivity as compared with overexpressed and tagged ILK. The AP-SISPROT technology and its combination with CRISPR Cas9 technology should be generally applicable for studying protein complexes in a more efficient and physiologically relevant manner.

Exposure of CCRF-CEM cells to acridone derivative 8a triggers tumor death via multiple mechanisms.

A newly synthesized acridone derivative 8a shows potent antitumor activity against CCRF-CEM leukemia cells. Herein, the first proteomic study of 8a effects in CCRF-CEM cells was performed by 2D nano-LC-ESI-MS/MS to better understand the mechanisms of action of 8a. Data analyses based on PLGS, STRING, Cytoscape, and database for annotation, visualization, and integrated discovery identified 55 proteins that were differentially expressed in response to 8a exposure. Multiple cellular pathways were affected, including chromatin organization, energy metabolism, DNA repair, oxidative-stress, and apoptosis. The changes in protein expression were further verified for PKM2. Moreover, 8a lowered down the expression of HEX and PFK-1. Lactate production was decreased in 8a-treated cells, indicating suppression of glycolysis. The elevated XRCC6 and decreased histone expression levels suggested increased DNA damage in 8a-treated cells, which was confirmed by the increased γ-H2AX foci. Molecular docking of 8a with DNA demonstrated direct interactions of 8a with DNA through three hydrogen bonds and four π-π interactions, potentially explaining the mode of action that 8a damaged to DNA. The differential protein profiling and dysfunction of metabolic pathways induced by 8a provide novel insights into the potential action mechanisms of 8a.

New benzimidazole acridine derivative induces human colon cancer cell apoptosis in vitro via the ROS-JNK signaling pathway.

AIM: To investigate the mechanisms underlying anticancer action of the benzimidazole acridine derivative N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine(8m) against human colon cancer cells in vitro. METHODS: Human colon cancer cell lines SW480 and HCT116 were incubated in the presence of 8m, and then the cell proliferation and apoptosis were measured. The expression of apoptotic/signaling genes and proteins was detected using RT-PCR and Western blotting. ROS generation and mitochondrial membrane depolarization were visualized with fluorescence microscopy. RESULTS: 8m dose-dependently suppressed the proliferation of SW480 and HCT116 cells with IC50 values of 6.77 and 3.33 µmol/L, respectively. 8m induced apoptosis of HCT116 cells, accompanied by down-regulation of Bcl-2, up-regulation of death receptor-5 (DR5), truncation of Bid, cleavage of PARP, and activation of caspases (including caspase-8 and caspase-9 as well as the downstream caspases-3 and caspase-7). Moreover, 8m selectively activated JNK and p38 without affecting ERK in HCT116 cells. Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis. In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells. Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells. CONCLUSION: The new benzimidazole acridine derivative, 8m exerts anticancer activity against human colon cancer cells in vitro by inducing both intrinsic and extrinsic apoptosis pathways via the ROS-JNK1 pathway.

Bifunctional HDAC and DNMT inhibitor induces viral mimicry activates the innate immune response in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a unique breast cancer subtype characterized by a lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. Since TNBC lacks ER, PR, and HER2, there are currently no drugs that specifically target TNBC. Therefore, the development of new drugs or effective treatment strategies to target TNBC has become an urgent clinical need. Research has shown that the application of histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors leads to genomic and epigenomic instability. This, in turn, triggers the activation of pattern recognition receptors (PRRs) and subsequently activates downstream interferon (IFN) signalling pathways. In this study, the bifunctional HDAC and DNMT inhibitor J208 exhibited antitumour activity in TNBC cell lines. J208 effectively induced apoptosis and cell cycle arrest at the G0/G1 phase, inhibiting cell migration and invasion in TNBC. Moreover, this bifunctional inhibitor induced the expression of endogenous retroviruses (ERVs) and elicited a viral mimicry response, which increased the intracellular levels of double-stranded RNA (dsRNA) to activate the innate immune signalling pathway in TNBC. In summary, we demonstrated that the bifunctional inhibitor J208, which is designed to inhibit HDAC and DNMT, has potent anticancer effects, providing a new research basis for reactivating antitumour immunity by triggering innate immune signalling and offering a promising strategy for TNBC treatment.

Dual inhibitors of DNMT and HDAC induce viral mimicry to induce antitumour immunity in breast cancer.

The existing conventional treatments for breast cancer, including immune checkpoint blockade, exhibit limited effects in some cancers, particularly triple-negative breast cancer. Epigenetic alterations, specifically DNMT and HDAC alterations, are implicated in breast cancer pathogenesis. We demonstrated that DNMTs and HDACs are overexpressed and positively correlated in breast cancer. The combination of DNMT and HDAC inhibitors has shown synergistic antitumour effects, and our previously designed dual DNMT and HDAC inhibitor (termed DNMT/HDACi) 15a potently inhibits breast cancer cell proliferation, migration, and invasion and induces apoptosis in vitro and in vivo. Mechanistically, 15a induces a viral mimicry response by promoting the expression of endogenous retroviral elements in breast cancer cells, thus increasing the intracellular level of double-stranded RNA to activate the RIG-I-MAVS pathway. This in turn promotes the production of interferons and chemokines and augments the expression of interferon-stimulated genes and PD-L1. The combination of 15a and an anti-PD-L1 antibody had an additive effect in vivo. These findings indicate that this DNMT/HDACi has immunomodulatory functions and enhances the effectiveness of immune checkpoint blockade therapy. A novel dual DNMT and HDAC inhibitor induces viral mimicry, which induces the accumulation of dsRNA to activate tumoral IFN signalling and cytokine production to enhance the immune response in breast cancer.

Novel dual inhibitors of PARP and HDAC induce intratumoral STING-mediated antitumor immunity in triple-negative breast cancer.

PARP inhibitors and HDAC inhibitors have been approved for the clinical treatment of malignancies, but acquired resistance of or limited effects on solid tumors with a single agent remain as challenges. Bioinformatics analyses and a combination of experiments had demonstrated the synergistic effects of PARP and HDAC inhibitors in triple-negative breast cancer. A series of novel dual PARP and HDAC inhibitors were rationally designed and synthesized, and these molecules exhibited high enzyme inhibition activity with excellent antitumor effects in vitro and in vivo. Mechanistically, dual PARP and HDAC inhibitors induced BRCAness to restore synthetic lethality and promoted cytosolic DNA accumulation, which further activates the cGAS-STING pathway and produces proinflammatory chemokines through type I IFN-mediated JAK-STAT pathway. Moreover, these inhibitors promoted neoantigen generation, upregulated antigen presentation genes and PD-L1, and enhanced antitumor immunity when combined with immune checkpoint blockade therapy. These results indicated that novel dual PARP and HDAC inhibitors have antitumor immunomodulatory functions in triple-negative breast cancer. Novel dual PARP and HDAC inhibitors induce BRCAness to restore synthetic lethality, activating tumoral IFN signaling via the cGAS-STING pathway and inducing cytokine production, promoting neoantigen generation and presentation to enhance the immune response.

Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-phenylurea derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor treatment.

Based on the roles of Raf1 and JNK1 in hepatocarcinoma development, scaffold-based drug design was employed to produce a series of compounds, which subsequently were synthesized and explored as potential dual inhibitors Raf1 and JNK1 kinases for anti-tumor treatment. The compound 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chloro-phenyl)urea (3d) showed 66%, 67% and 13% inhibition rate at 50 µM against Raf1, JNK1 and p38-alpha, respectively, but no effect on ERK1 and ERK2, and inhibited the expression of pERK1/2 markedly and HepG2 cells proliferation with IC(50) at 8.3 µM. Furthermore, 3d showed lower toxicity against normal liver cell-lines QSG7701 and HL7702. Molecular docking study further showed that 3d can fit into binding domain of JNK1 and Raf1. Our data suggested the activities of 3d were associated with dual inhibition of JNK1 and Raf1 kinases.

Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression.

Androgen receptor (AR) signaling plays important roles in breast cancer progression. We show here that Kindlin-2, a focal adhesion protein, is critically involved in the promotion of AR signaling and breast cancer progression. Kindlin-2 physically associates with AR and Src through its two neighboring domains, namely F1 and F0 domains, resulting in formation of a Kindlin-2-AR-Src supramolecular complex and consequently facilitating Src-mediated AR Tyr-534 phosphorylation and signaling. Depletion of Kindlin-2 was sufficient to suppress Src-mediated AR Tyr-534 phosphorylation and signaling, resulting in diminished breast cancer cell proliferation and migration. Re-expression of wild-type Kindlin-2, but not AR-binding-defective or Src-binding-defective mutant forms of Kindlin-2, in Kindlin-2-deficient cells restored AR Tyr-534 phosphorylation, signaling, breast cancer cell proliferation and migration. Furthermore, re-introduction of phosphor-mimic mutant AR-Y534D, but not wild-type AR reversed Kindlin-2 deficiency-induced inhibition of AR signaling and breast cancer progression. Finally, using a genetic knockout strategy, we show that ablation of Kindlin-2 from mammary tumors in mouse significantly reduced AR Tyr-534 phosphorylation, breast tumor progression and metastasis in vivo. Our results suggest a critical role of Kindlin-2 in promoting breast cancer progression and shed light on the molecular mechanism through which it functions in this process.

Synergistic optimization of Liquid Chromatography and Mass Spectrometry parameters on Orbitrap Tribrid mass spectrometer for high efficient data-dependent proteomics.

Steady improvement in Orbitrap-based mass spectrometry (MS) technologies has greatly advanced the peptide sequencing speed and depth. In-depth analysis of the performance of state-of-the-art MS and optimization of key parameters can improve sequencing efficiency. In this study, we first systematically compared the performance of two popular data-dependent acquisition approaches, with Orbitrap as the first-stage (MS1) mass analyzer and the same Orbitrap (high-high approach) or ion trap (high-low approach) as the second-stage (MS2) mass analyzer, on the Orbitrap Fusion mass spectrometer. High-high approach outperformed high-low approach in terms of better saturation of the scan cycle and higher MS2 identification rate. However, regardless of the acquisition method, there are still more than 60% of peptide features untargeted for MS2 scan. We then systematically optimized the MS parameters using the high-high approach. Increasing the isolation window in the high-high approach could facilitate faster scan speed, but decreased MS2 identification rate. On the contrary, increasing the injection time of MS2 scan could increase identification rate but decrease scan speed and the number of identified MS2 spectra. Dynamic exclusion time should be set properly according to the chromatography peak width. Furthermore, we found that the Orbitrap analyzer, rather than the analytical column, was easily saturated with higher loading amount, thus limited the dynamic range of MS1-based quantification. By using optimized parameters, 10 000 proteins and 110 000 unique peptides were identified by using 20 h of effective liquid chromatography (LC) gradient time. The study therefore illustrated the importance of synchronizing LC-MS precursor ion targeting, fragment ion detection, and chromatographic separation for high efficient data-dependent proteomics.

Phosphorylation-regulated HMGA1a-P53 interaction unveils the function of HMGA1a acidic tail phosphorylations via synthetic proteins.

As a typical member of intrinsically disordered proteins (IDPs), HMGA1a carries many post-translational modifications (PTMs). To study the undefined function of acidic tail phosphorylations, seven HMGA1a proteins with site-specific modification(s) were chemically synthesized via Ser/Thr ligation. We found that the phosphorylations significantly inhibit HMGA1a-P53 interaction and the phosphorylations can induce conformational change of HMGA1a from an "open state" to a "close state." Notably, the positively charged lysine-arginine (KR) clusters are responsible for modulating HMGA1a conformation via electrostatic interaction with the phosphorylated acidic tail. Finally, we used a synthetic protein-affinity purification mass spectrometry (SP-AP-MS) methodology to profile the specific interactors, which further supported the function of HMGA1a phosphorylation. Collectively, this study highlights a mechanism for regulating IDPs' conformation and function by phosphorylation of non-protein-binding domain and showcases that the protein chemical synthesis in combination with mass spectrometry can serve as an efficient tool to study the IDPs' PTMs.

Design, synthesis and evaluation of novel ErbB/HDAC multitargeted inhibitors with selectivity in EGFRT790M mutant cell lines.

Acquired resistance leads to the failure of EGFR TKIs in NSCLC treatment. A novel series of hydroxamic acid-containing 4-aminoquinazoline derivatives as irreversible ErbB/HDAC multitargeted inhibitors for NSCLC therapy had been designed and synthesized, which displayed weak anti-proliferative activity in several EGFR wild-type cancer cell lines (NCI-H838, SK-BR-3, A549, A431) yet retained moderate activity to EGFRT790M resistance mutation harboring NCI-H1975 cells. The mechanistic studies revealed that the representative compound 11e was able to inhibit the phosphorylation of EGFR, up-regulate hyperacetylation of histone H3 and even reduce the expression of EGFR and Akt in NCI-H1975 cells. In further assays, compound 11e also showed moderate anti-proliferative activity in other EGFRT790M harboring tumor cell lines (NCI-H820, Ba/F3_EGFR_Del19-T790M-C797S) and low toxicities in normal cell lines (HL-7702, FHC). This selectivity of designed multitargeted compounds could serve as a potential strategy to circumvent multiple mechanisms of acquired resistance to EGFR-targeted therapy without severe toxicities and side effects resulting from broad inhibition.

Design, synthesis and anti-tumor evaluation of 1,2,4-triazol-3-one derivatives and pyridazinone derivatives as novel CXCR2 antagonists.

Chemokine receptor 2 (CXCR2) is the receptor of glutamic acid-leucine-arginine sequence-contained chemokines CXCs (ELR+ CXCs). In recent years, CXCR2-target treatment strategy has come a long way in cancer therapy. CXCR2 antagonists could block CXCLs/CXCR2 axis, and are widely used in regulating immune cell migration, tumor metastasis, apoptosis and angiogenesis. Herein, two series of new CXCR2 small-molecule inhibitors, including 1,2,4-triazol-3-one derivatives 1-11 and pyridazinone derivatives 12-22 were designed and synthesized based on the proof-to-concept. The pyridazinone derivative 18 exhibited good CXCR2 antagonistic activity (69.4 ± 10.5 %Inh at 10 µM) and demonstrated its significant anticancer metastasis activity in MDA-MB-231 cells and remarkable anti-angiogenesis activity in HUVECs. Furthermore, noteworthy was that 18 exhibited an obvious synergistic effect with Sorafenib in anti-proliferation assay in MDA-MB-231 cells. Moreover, 18 showed a distinct reduction of the phosphorylation levels of both PI3K and AKT proteins in MDA-MB-231 cells, and also affected the expression levels of other PI3K/AKT signaling pathway-associated proteins. The molecular docking studies of 18 with CXCR2 also verified the rationality of our design strategy. All of these results revealed pyridazinone derivative 18 as a promising CXCR2 antagonist for future cancer therapy.