An in-line method for high-throughput screening of protein tyrosine phosphatase receptor type O inhibitors by capillary electrophoresis based on electrophoretically mediated microanalysis.

Protein tyrosine phosphatase receptor type O (PTPRO) plays an important role in inflammation-related pathways and has become an emerging drug target. In this study, we developed an in-line capillary electrophoresis (CE) method for the investigation of the enzymatic activity of PTPRO, which was based on electrophoretically mediated microanalysis (EMMA). After a thorough method validation of the optimized conditions, this protocol was successfully employed to determine the kinetics of PTPRO as well as the half-maximal inhibitory concentration (IC50) of two typical PTPRO inhibitors. The final results were consistent with the values obtained through classical ultraviolet-visible (UV-vis) spectrophotometry. Our new method exhibited improved accuracy and reduced consumption, avoiding the disadvantages of traditional methods. This work provides a new strategy for PTPRO enzyme kinetic studies as well as inhibitory activity determination through capillary electrophoresis for the first time.

Deciphering the Allosteric Activation Mechanism of SIRT6 Using Molecular Dynamics Simulations.

As a member of the histone deacetylase protein family, the NAD+-dependent SIRT6 plays an important role in maintaining genomic stability and regulating cell metabolism. Interestingly, SIRT6 has been found to have a preference for hydrolyzing long-chain fatty acyls relative to deacetylation, and it can be activated by fatty acids. However, the mechanisms by which SIRT6 recognizes different substrates and can be activated by small molecular activators are still not well understood. In this study, we carried out extensive molecular dynamic simulations to shed light on these mechanisms. Our results revealed that the binding of the myristoylated substrate stabilizes the catalytically favorable conformation of NAD+, while the binding of the acetyl-lysine substrate leads to a loose binding of NAD+ in SIRT6. Based on these observations, we proposed a reasonable allosteric binding mode for myristic acid, which can enhance the catalytic activity of SIRT6 by stabilizing the binding of NAD+ with His131 as well as the acetylated substrate. Furthermore, our molecular dynamics simulations demonstrated that synthetic SIRT6 activators, such as UBCS039, MDL-801, and 12q, block the flipping of ribose in NAD+ and therefore can stabilize substrate-NAD+-His131 interactions in a manner similar to fatty acids. In summary, our newly proposed activation mechanism of SIRT6 highlights the importance of protein-substrate interactions, which would facilitate the rational design of new SIRT6 activators.

Targeting histone deacetylases for cancer therapy: Trends and challenges.

Dysregulation of histone deacetylases (HDACs) is closely related to tumor development and progression. As promising anticancer targets, HDACs have gained a great deal of research interests and two decades of effort has led to the approval of five HDAC inhibitors (HDACis). However, currently traditional HDACis, although effective in approved indications, exhibit severe off-target toxicities and low sensitivities against solid tumors, which have urged the development of next-generation of HDACi. This review investigates the biological functions of HDACs, the roles of HDACs in oncogenesis, the structural features of different HDAC isoforms, isoform-selective inhibitors, combination therapies, multitarget agents and HDAC PROTACs. We hope these data could inspire readers with new ideas to develop novel HDACi with good isoform selectivity, efficient anticancer effect, attenuated adverse effect and reduced drug resistance.

Discovery of benzofuran-2-carboxylic acid derivatives as lymphoid tyrosine phosphatase (LYP) inhibitors for cancer immunotherapy.

Lymphoid-tyrosine phosphatase (LYP) is mainly expressed in the immune system and plays an important role in the T-cell receptor (TCR) signaling pathway and tumor immunity. Herein, we identify benzofuran-2-carboxylic acid as a potent pTyr mimic and design a new series of new LYP inhibitors. The most active compound, D34 and D14, reversibly inhibits LYP (Ki = 0.93 µM and 1.34 µM) and possess a certain degree of selectivity toward other phosphatases. Meanwhile, D34 and D14 regulate the TCR signaling by specifically inhibiting LYP. In particular, D34 and D14 significantly suppress tumor growth in an MC38 syngeneic mouse model by boosting antitumor immunity, including activation of T-cell and inhibition of M2 macrophage polarization. Moreover, treatment of D34 or D14 upregulate PD-1/PD-L1 expression, which can be leveraged with PD-1/PD-L1 inhibition to augment immunotherapy. In summary, our study demonstrates the feasibility of targeting LYP for cancer immunotherapy and provides new lead compounds for further drug development.

Characterization of an allosteric inhibitor of fungal-specific C-24 sterol methyltransferase to treat Candida albicans infections.

Filamentation is an important virulence factor of the pathogenic fungus Candida albicans. The abolition of Candida albicans hyphal formation by disrupting sterol synthesis is an important concept for the development of antifungal drugs with high safety. Here, we conduct a high-throughput screen using a C. albicans strain expressing green fluorescent protein-labeled Dpp3 to identify anti-hypha agents by interfering with ergosterol synthesis. The antipyrine derivative H55 is characterized to have minimal cytotoxicity and potent inhibition of C. albicans hyphal formation in multiple cultural conditions. H55 monotherapy exhibits therapeutic efficacy in mouse models of azole-resistant candidiasis. H55 treatment increases the accumulation of zymosterol, the substrate of C-24 sterol methyltransferase (Erg6). The results of enzyme assays, photoaffinity labeling, molecular simulation, mutagenesis, and cellular thermal shift assays support H55 as an allosteric inhibitor of Erg6. Collectively, H55, an inhibitor of the fungal-specific enzyme Erg6, holds potential to treat C. albicans infections.

Design, synthesis and bioactivity evaluations of 8-substituted-quinoline-2-carboxamide derivatives as novel histone deacetylase (HDAC) inhibitors.

The inhibition of histone deacetylases (HDACs) has been considered a promising therapeutic strategy for treatment of many diseases, especially cancer. In the current study, a series of 8-substituted quinoline-2-carboxamide derivatives were designed and synthesized as potent HDAC inhibitors. The most potent compound 21 g (IC50 = 0.050 µM) exhibited 3-fold greater HDAC inhibitory activity compared to the known HDAC inhibitor Vorinostat (IC50 = 0.137 µM). Additionally, compound 21g exhibited low toxicity against normal cells(IC50 in HUVEC cell > 50 µM) and showed good liver microsomal stability, therefore, may serve as a new lead compound for further development.

Structural insight into TIPE1 functioning as a lipid transfer protein.

As a member of the tumor necrosis factor-α-induced protein 8 (TNFAIP8/TIPE) family, TIPE1 has been found to be associated with many cellular signaling pathways in regulating apoptosis, autophagy, and tumorigenesis. However, the position of TIPE1 in the signaling network remains elusive. Here we present the crystal structure of zebrafish TIPE1 in complex with phosphatidylethanolamine (PE) at a resolution of 1.38 Å. By comparison with structures of other three TIPE family proteins, a universal phospholipid-binding mode was proposed. Namely, the hydrophobic cavity binds to fatty acid tails, while 'X-R-R' triad nearby the entrance of cavity recognizes the phosphate group head. Using molecular dynamics (MD) simulations, we further elaborated the mechanism of how the lysine-rich N-terminal domain assisting TIPE1 to favorably bind to phosphatidylinositol (PI). Beside small molecule substrate, we identified Gαi3 as a direct-binding partner of TIPE1 using GST pull-down assay and size-exclusion chromatography. Analyses of key-residue mutations and predicted complex structure revealed that the binding mode of TIPE1 to Gαi3 could be non-canonical. In summary, our findings narrowed down TIPE1's position in Gαi3-related and PI-inducing signaling pathways.Communicated by Ramaswamy H. Sarma.

Interaction of PKR with STCS1: an indispensable step in the biosynthesis of lunularic acid in Marchantia polymorpha.

Unlike bibenzyls derived from the vascular plants, lunularic acid (LA), a key precursor for macrocyclic bisbibenzyl synthesis in nonvascular liverworts, exhibits the absence of one hydroxy group within the A ring. It was hypothesized that both polyketide reductase (PKR) and stilbenecarboxylate synthase 1 (STCS1) were involved in the LA biosynthesis, but the underlined mechanisms have not been clarified. This study used bioinformatics analysis with molecular, biochemical and physiological approaches to characterize STCS1s and PKRs involved in the biosynthesis of LA. The results indicated that MpSTCS1s from Marchantia polymorpha catalyzed both C2→C7 aldol-type and C6→C1 Claisen-type cyclization using dihydro-p-coumaroyl-coenzyme A (CoA) and malonyl-CoA as substrates to yield a C6-C2-C6 skeleton of dihydro-resveratrol following decarboxylation and the C6-C3-C6 type of phloretin in vitro. The protein-protein interaction of PKRs with STCS1 (PPI-PS) was revealed and proved essential for LA accumulation when transiently co-expressed in Nicotiana benthamiana. Moreover, replacement of the active domain of STCS1 with an 18-amino-acid fragment from the chalcone synthase led to the PPI-PS greatly decreasing and diminishing the formation of LA. The replacement also increased the chalcone formation in STCS1s. Our results highlight a previously unrecognized PPI in planta that is indispensable for the formation of LA.

Development of QSRR model for hydroxamic acids using PCA-GA-BP algorithm incorporated with molecular interaction-based features.

As a potent zinc chelator, hydroxamic acid has been applied in the design of inhibitors of zinc metalloenzyme, such as histone deacetylases (HDACs). A series of hydroxamic acids with HDAC inhibitory activities were subjected to the QSRR (Quantitative Structure-Retention Relationships) study. Experimental data in combination with calculated molecular descriptors were used for the development of the QSRR model. Specially, we employed PCA (principal component analysis) to accomplish dimension reduction of descriptors and utilized the principal components of compounds (16 training compounds, 4 validation compounds and 7 test compounds) to execute GA (genetic algorithm)-BP (error backpropagation) algorithm. We performed double cross-validation approach for obtaining a more convincing model. Moreover, we introduced molecular interaction-based features (molecular docking scores) as a new type of molecular descriptor to represent the interactions between analytes and the mobile phase. Our results indicated that the incorporation of molecular interaction-based features significantly improved the accuracy of the QSRR model, (R2 value is 0.842, RMSEP value is 0.440, and MAE value is 0.573). Our study not only developed QSRR model for the prediction of the retention time of hydroxamic acid in HPLC but also proved the feasibility of using molecular interaction-based features as molecular descriptors.

Inhibition of fungal pathogenicity by targeting the H2S-synthesizing enzyme cystathionine ß-synthase.

The global emergence of antifungal resistance threatens the limited arsenal of available treatments and emphasizes the urgent need for alternative antifungal agents. Targeting fungal pathogenic functions is an appealing alternative therapeutic strategy. Here, we show that cystathionine ß-synthase (CBS), compared with cystathionine γ-lyase, is the major enzyme that synthesizes hydrogen sulfide in the pathogenic fungus Candida albicans. Deletion of CBS leads to deficiencies in resistance to oxidative stress, retarded cell growth, defective hyphal growth, and increased ß-glucan exposure, which, together, reduce the pathogenicity of C. albicans. By high-throughput screening, we identified protolichesterinic acid, a natural molecule obtained from a lichen, as an inhibitor of CBS that neutralizes the virulence of C. albicans and exhibits therapeutic efficacy in a murine candidiasis model. These findings support the application of CBS as a potential therapeutic target to fight fungal infections.

Structural basis of human SNAPc recognizing proximal sequence element of snRNA promoter.

In eukaryotes, small nuclear RNAs (snRNAs) function in many fundamental cellular events such as precursor messenger RNA splicing, gene expression regulation, and ribosomal RNA processing. The snRNA activating protein complex (SNAPc) exclusively recognizes the proximal sequence element (PSE) at snRNA promoters and recruits RNA polymerase II or III to initiate transcription. In view that homozygous gene-knockout of SNAPc core subunits causes mouse embryonic lethality, functions of SNAPc are almost housekeeping. But so far, the structural insight into how SNAPc assembles and regulates snRNA transcription initiation remains unclear. Here we present the cryo-electron microscopy structure of the essential part of human SNAPc in complex with human U6-1 PSE at an overall resolution of 3.49 Å. This structure reveals the three-dimensional features of three conserved subunits (N-terminal domain of SNAP190, SNAP50, and SNAP43) and explains how they are assembled into a stable mini-SNAPc in PSE-binding state with a "wrap-around" mode. We identify three important motifs of SNAP50 that are involved in both major groove and minor groove recognition of PSE, in coordination with the Myb domain of SNAP190. Our findings further elaborate human PSE sequence conservation and compatibility for SNAPc recognition, providing a clear framework of snRNA transcription initiation, especially the U6 system.

Discovery of Novel Src Homology-2 Domain-Containing Phosphatase 2 and Histone Deacetylase Dual Inhibitors with Potent Antitumor Efficacy and Enhanced Antitumor Immunity.

Both Src homology-2 domain-containing phosphatase 2 (SHP2) and histone deacetylase (HDAC) are important oncoproteins and potential immunomodulators. In this study, we first observed a synergistic antiproliferation effect of an allosteric SHP2 inhibitor (SHP099) and HDAC inhibitor (SAHA) in MV4-11 cells. Inspired by this result, a series of SHP2/HDAC dual inhibitors were designed based on the pharmacophore fusion strategy. Among these inhibitors, the most potent compound 8t showed excellent inhibitory activities against SHP2 (IC50 = 20.4 nM) and HDAC1 (IC50 = 25.3 nM). In particular, compound 8t exhibited improved antitumor activities compared with those of SHP099 and SAHA in vitro and in vivo. Our study also indicated that treatment with 8t could trigger efficient antitumor immunity by activating T cells, enhancing the antigen presentation function and promoting cytokine secretion. To our knowledge, we report the first small molecular SHP2/HDAC dual inhibitor and demonstrate a new strategy for cancer immunotherapy.

Design, synthesis and biological evaluations of novel farnesoid X receptor (FXR) agonists.

As a member of the nuclear receptor superfamily, the farnesoid X receptor (FXR) is a bile acid activated transcription factor. FXR is involved in many important metabolic processes and serves as a promising therapeutic target for nonalcoholic steatohepatitis (NASH). Since discovered, the first non-steroidal FXR agonist GW4064 has been widely used to explore the biological functions of FXR, however, the low pharmacokinetic limited its further clinical application. In current study, we designed a series of substituted isothiazoles as new FXR agonists. Among them, five compounds exhibited better FXR agonistic activity than GW4064. Specially, the most potent compound S5 possessed better pharmacokinetic profile and in vivo potency than lead compound.

Structure-Based Design of 2-Aminopurine Derivatives as CDK2 Inhibitors for Triple-Negative Breast Cancer.

Cyclin-dependent kinase 2 (CDK2) regulates the progression of the cell cycle and is critically associated with tumor growth. Selective CDK2 inhibition provides a potential therapeutic benefit against certain tumors. Purines and related heterocycle (e.g., R-Roscovitine) are important scaffolds in the development of CDK inhibitors. Herein, we designed a new series of 2-aminopurine derivatives based on the fragment-centric pocket mapping analysis of CDK2 crystal structure. Our results indicated that the introduction of polar substitution at the C-6 position of purine would be beneficial for CDK2 inhibition. Among them, compound 11l showed good CDK2 inhibitory activity (IC50 = 19 nM) and possessed good selectivity against other CDKs. Further in vitro tests indicated that compound 11l possesses anti-proliferation activity in triple-negative breast cancer (TNBC) cells. Moreover, molecular dynamics simulation suggested the favorable binding mode of compound 11l, which may serve as a new lead compound for the future development of CDK2 selective inhibitors.

Design, synthesis and biological evaluation of hydantoin derivatives as Mcl-1 selective inhibitors.

As a member of Bcl-2 protein family, myeloid cell leukemia-1 (Mcl-1) plays a critical role in cell apoptosis and has become a promising anti-cancer drug target. Herein, we designed and synthesized a series of hydantoin derivatives as novel Mcl-1 inhibitors based on our previously developed lead compound. Among them, compound M23 and M24 exhibited good binding affinities against Mcl-1 with Ki values of 0.49 µM and 0.33 µM respectively. Especially, compound M23 exhibited good selectivity over Bcl-xL, whereas compound M24 possessed good selectivity over both Bcl-2 and Bcl-xL. Furthermore, we also investigated the effects of these new Mcl-1 inhibitors on cell proliferation, apoptosis and mitochondrial membrane potential, as well as the stability in plasma.

Discovery of DNA-Targeting HDAC Inhibitors with Potent Antitumor Efficacy In Vivo That Trigger Antitumor Immunity.

We observed a synergistic antiproliferation effect with combined use of a DNA minor groove binder and a histone deacetylase (HDAC) inhibitor. Inspired by this result, a new series of benzimidazole-hydroxamate hybrids were designed and synthesized to target both DNA minor groove and HDAC. The most active compounds 9k and 9l not only exhibited improved HDAC inhibitory activities compared to SAHA but also possessed potent antiproliferation activities against tumor cells. Importantly, compounds 9k and 9l showed good in vivo antitumor efficacies in both HEL xenograft model and murine melanoma model. We also found that 9k and 9l promote the antigen presentation and activate T cells, thereby triggering antitumor immunity. Moreover, these inhibitors reshaped the tumor immune microenvironment by inhibiting the recruitment of Treg cells and promoting the polarization of tumor-infiltrating macrophages to M2 type with antitumor activity. Our study validated the effectiveness of incorporating a DNA-binding fragment in HDAC inhibitors as novel multitargeting antitumor agents.

Palmarumycin P3 Reverses Mrr1-Mediated Azole Resistance by Blocking the Efflux Pump Mdr1.

Palmarumycin P3 (PP3) reduces fluconazole-induced MDR1 transcription to reverse azole resistance in clinical Candida strains. Here, we demonstrated that PP3 restores the susceptibility to several antifungal drugs for Candida albicans strains with gain-of-function mutations in the transcription factor Mrr1. In addition, PP3 inhibits the efflux of Mdr1 substrates by C. albicans strains harboring hyperactive MRR1 alleles. Molecular docking revealed that PP3 is a potential Mdr1 blocker that binds to the substrate binding pocket of Mdr1.

Boronate-Based Fluorescent Probes as a Prominent Tool for H2O2 Sensing and Recognition.

Given the crucial association of hydrogen peroxide with a wide range of human diseases, this compound has currently earned the reputation of being a popular biomolecular target. Although various analytical methods have attracted our attention, fluorescent probes have been used as prominent tools to determine H2O2 to reflect the physiological and pathological conditions of biological systems. The sensitive responsive part of these probes is the boronate ester and boronic acid groups, which are important reporters for H2O2 recognition. In this review, we summarize boronate ester/boronic acid group-based fluorescent probes for H2O2 reported from 2012 to 2020, and we have generally classified the fluorophores into six categories to exhaustively elaborate the design strategy and comprehensive systematic performance. We hope that this review will inspire the exploration of new fluorescent probes based on boronate ester/boronic acid groups for the detection of H2O2 and other relevant analytes.

Recent advances in the development of allosteric protein tyrosine phosphatase inhibitors for drug discovery.

Protein tyrosine phosphatases (PTPs) superfamily catalyzes tyrosine de-phosphorylation which affects a myriad of cellular processes. Imbalance in signal pathways mediated by PTPs has been associated with development of many human diseases including cancer, metabolic, and immunological diseases. Several compelling evidence suggest that many members of PTP family are novel therapeutic targets. However, the clinical development of conventional PTP-based active-site inhibitors originally was hampered by the poor selectivity and pharmacokinetic properties. In this regard, PTPs has been widely dismissed as "undruggable." Nonetheless, allosteric modulation has become increasingly an influential and alternative approach that can be exploited for drug development against PTPs. Unlike active-site inhibitors, allosteric inhibitors exhibit a remarkable target-selectivity, drug-likeness, potency, and in vivo activity. Intriguingly, there has been a high interest in novel allosteric PTPs inhibitors within the last years. In this review, we focus on the recent advances of allosteric inhibitors that have been explored in drug discovery and have shown an excellent result in the development of PTPs-based therapeutics. A special emphasis is placed on the structure-activity relationship and molecular mechanistic studies illustrating applications in chemical biology and medicinal chemistry.

Potential applications of BPFP1 in Bcl-2 protein quantification, carcinoma cell visualization, cell sorting and early cancer diagnosis.

Overexpression of the Bcl-2 protein has emerged as a hallmark of carcinoma cells and can be employed as a biochemical biomarker of these cells. Therefore, some Bcl-2 protein fluorescence probes (BPFPs) were designed for Bcl-2 protein quantification and carcinoma cells labeling. The high Bcl-2 protein binding affinity (Ki < 1 nM) and selectivity (over 50,000-fold Bcl-2 protein selectivity against Mcl-1 protein) of BPFP1 endow it with the ability to detect trace amounts of Bcl-2 protein. After being incubated with a range of concentrations of Bcl-2 protein, BPFP1 exhibited the desired fluorescence properties and its fluorescence intensity is proportional to Bcl-2 protein concentration. Therefore, BPFP1 provides a convenient approach for Bcl-2 protein quantification and we could determine the concentration of Bcl-2 protein based on the BPFP1's fluorescence intensity. Subsequent studies revealed that BPFP1 can fluorescently label carcinoma cells by binding to overexpressed Bcl-2 protein in living cells, and can distinguish carcinoma cells (HL-60 cells and ACHN cells) from normal-tissue cells (HUVECs) according to the different Bcl-2 protein expression levels between carcinoma cells and normal tissue cells. In the present study, BPFP1 represents a new tool for Bcl-2 protein quantification, carcinoma cell visualization and cell sorting. Moreover, BPFP1 can be used in the future for early cancer diagnosis by detecting carcinoma cells in patient tissues.