An affinity prediction approach for the ligand of E3 ligase Cbl-b and an insight into substrate binding pattern.

Protein-protein interactions (PPIs) are essentially fundamental to all cellular processes, so that developing small molecule inhibitors of PPIs have great significance despite representing a huge challenge. Studying PPIs with the help of peptide motifs could obtain the structural information and reference significance to reduce the difficulty in the development of small molecules. Computational methods are powerful tools to characterize peptide-protein interactions, especially molecular dynamics simulation and binding free energy calculation. Here, we established an affinity prediction model suitable for Casitas B lymphoma-b (Cbl-b) and phosphorylated motif system. According to the affinity data set of multiple truncated peptides, the force field, solvent model, and internal dielectric constant of molecular mechanics/generalized Born surface area (MM/GBSA) method were optimized. Further, we predicted the affinity of the rationally designed new sequences through this model and obtained a new 6-mer motif with a 7-fold increase in affinity and the comprehensive structure-activity relationship. Moreover, we proposed an insight of unexpected activity of the truncated 5-mer peptide and revealed the possible binding mode of the new highly active 6-mer motif by extended simulation. Our results showed that the activity enhancement of the truncated peptide was caused by the acetyl-mediated conformation change. The side chain of Arg and pTyr in the 6-mer motif co-occupied the site p1 to form numerous hydrogen bond interactions and increased hydrophobic interaction formed with Tyr266, leading to the higher affinity. The present work provided a reference to investigate the PPI of Cbl-b and phosphorylated substrates and guided the development of Cbl-b inhibitors.

Design, synthesis, and initial evaluation of affinity-based small molecular probe for detection of WDR5.

WDR5, a subunit of the SET/MLL complex, plays critical roles in various biological progresses and are abnormally expressed in many cancers. Here we report the design, synthesis, and biochemical characterization of a new chemical tool to capture WDR5 protein. The probe is a biotinylated version of compound 30 that is a potent WDR5 inhibitor we previously reported. Importantly, the probe displayed high affinity to WDR5 protein in vitro binding potency and showed the ability in specifically and real time monitoring WDR5 protein. Further, the biotinylated tag of the probe enabled selectively "chemoprecipitation" of WDR5 from whole cell lysates of MV4-11. This probe provided a new approach to identify the overexpressed WDR5 protein in different cancer cells and applications to proteomic analysis of WDR5 and WDR5-binding partners.

Optimization and bioevaluation of Cdc37-derived peptides: An insight into Hsp90-Cdc37 protein-protein interaction modulators.

Targeting Hsp90-Cdc37 protein-protein interaction (PPI) is becoming an alternative approach for future anti-cancer drug development. We previously reported the discovery of an eleven-residue peptide (Pep-1) with micromolar activity for the disruption of Hsp90-Cdc37 PPI. Efforts to improve upon the Pep-1 led to the discovery of more potent modulators for Hsp90-Cdc37 PPI. Through the analysis of peptides binding patterns, more peptides were designed for further verification which resulted in Pep-5, the shortest peptide targeting Hsp90-Cdc37, exerting the optimal structure and the most efficient binding mode. Subsequent MD simulation analysis also confirmed that Pep-5 could perform more stable binding ability and better ligand properties than Pep-1. Under the premise of retentive binding capacity, Pep-5 exhibited lower molecular weight and higher ligand efficiency with a Kd value of 5.99µM (Pep-1 Kd=6.90µM) in both direct binding determination and biological evaluation. The optimal and shortest Pep-5 might provide a breakthrough and a better model for the future design of small molecule inhibitors targeting Hsp90-Cdc37 PPI.

The Keap1-Nrf2-ARE Pathway As a Potential Preventive and Therapeutic Target: An Update.

The Keap1-Nrf2-ARE ((Kelch-like ECH-Associating protein 1) nuclear factor erythroid 2 related factor 2-antioxidant response element) pathway is one of the most important defense mechanisms against oxidative and/or electrophilic stresses, and it is closely associated with inflammatory diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and aging. In recent years, progress has been made in strategies aimed at modulating the Keap1-Nrf2-ARE pathway. The Nrf2 activator DMF (Dimethylfumarates) has been approved by the FDA as a new first-line oral drug to treat patients with relapsing forms of multiple sclerosis, while a phase 3 study of another promising candidate, CDDO-Me, was terminated for safety reasons. Directly inhibiting Keap1-Nrf2 protein-protein interactions as a novel Nrf2-modulating strategy has many advantages over using electrophilic Nrf2 activators. The development of Keap1-Nrf2 protein-protein interaction inhibitors has become a topic of intense research, and potent inhibitors of this target have been identified. In addition, inhibiting Nrf2 activity has attracted an increasing amount of attention because it may provide an alternative cancer therapy. This review summarizes the molecular mechanisms and biological functions of the Keap1-Nrf2-ARE system. The main focus of this review is on recent progress in studies of agents that target the Keap1-Nrf2-ARE pathway and the therapeutic applications of such agents.

Molecular similarity guided optimization of novel Nrf2 activators with 1,2,4-oxadiazole core.

DDO-7204 is a novel Nrf2 activator first identified through screening of in-house database by ARE-luciferase reporter gene assay. To further optimize this kind of Nrf2 activators efficiently, the hit-based substructure search was applied to screen the Specs database virtually. DDO-7204 contains three rings of A, B, C. SAR results showed that: for ring A, the cyclane substituent is beneficial for ARE inductivity. Enhanced flexibility of linker between ring A and ring B is not preferable for the Nrf2 activity. Ring A replaced by heterocyclic aromatic is beneficial for the Nrf2 activity. The resulting compound 7 was more potent than DDO-7204. Compound 7 can induce Nrf2 translocation into nuclear not only in HCT116 cells, but also in three normal cells such as L02, NCM460 and PC12 cells. The Nrf2-regualted genes, γ-GCS, NQO1 and HO-1, were up-regulated at a concentration-dependent manner. In addition, compound 7 showed cytoprotective effects on the three normal cells against the damage of H2O2.

Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis.

E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

Integrated analysis of tertiary lymphoid structures and immune infiltration in ccRCC microenvironment revealed their clinical significances: a multicenter cohort study.

BACKGROUND: Tertiary lymphoid structures (TLSs) serve as organized lymphoid aggregates that influence immune responses within the tumor microenvironment. This study aims to investigate the characteristics and clinical significance of TLSs and tumor-infiltrating lymphocytes (TILs) in clear cell renal cell carcinoma (ccRCC). METHODS: TLSs and TILs were analyzed comprehensively in 754 ccRCC patients from 6 academic centers and 532 patients from The Cancer Genome Atlas. Integrated analysis was performed based on single-cell RNA-sequencing datasets from 21 ccRCC patients to investigate TLS heterogeneity in ccRCC. Immunohistochemistry and multiplex immunofluorescence were applied. Cox regression and Kaplan-Meier analyses were used to reveal the prognostic significance. RESULTS: The study demonstrated the existence of TLSs and TILs heterogeneities in the ccRCC microenvironment. TLSs were identified in 16% of the tumor tissues in 113 patients. High density (>0.6/mm2) and maturation of TLSs predicted good overall survival (OS) (p<0.01) in ccRCC patients. However, high infiltration (>151) of scattered TILs was an independent risk factor of poor ccRCC prognosis (HR=14.818, p<0.001). The presence of TLSs was correlated with improved progression-free survival (p=0.002) and responsiveness to therapy (p<0.001). Interestingly, the combination of age and TLSs abundance had an impact on OS (p<0.001). Higher senescence scores were detected in individuals with immature TLSs (p=0.003). CONCLUSIONS: The study revealed the contradictory features of intratumoral TLSs and TILs in the ccRCC microenvironment and their impact on clinical prognosis, suggesting that abundant and mature intratumoral TLSs were associated with decreased risks of postoperative ccRCC relapse and death as well as favorable therapeutic response. Distinct spatial distributions of immune infiltration could reflect effective antitumor or protumor immunity in ccRCC.

Discovery of Pyrazolo[1,5-a]pyrimidine Derivative as a Novel and Selective ALKBH5 Inhibitor for the Treatment of AML.

M6A (N6-methyladenosine) plays a significant role in regulating RNA processing, splicing, nucleation, translation, and stability. AlkB homologue 5 (ALKBH5) is an Fe(II)/2-oxoglutarate (2-OG)-dependent dioxygenase that demethylates mono- or dimethylated adenosines. ALKBH5 can be regarded as an oncogenic factor for various human cancers. However, the discovery of potent and selective ALKBH5 inhibitors remains a challenge. We identified DDO-2728 as a novel and selective inhibitor of ALKBH5 by structure-based virtual screening and optimization. DDO-2728 was not a 2-oxoglutarate analogue and could selectively inhibit the demethylase activity of ALKBH5 over FTO. DDO-2728 increased the abundance of m6A modifications in AML cells, reduced the mRNA stability of TACC3, and inhibited cell cycle progression. Furthermore, DDO-2728 significantly suppressed tumor growth in the MV4-11 xenograft mouse model and showed a favorable safety profile. Collectively, our results highlight the development of a selective probe for ALKBH5 that will pave the way for the further study of ALKBH5 targeting therapies.

Development of Orally Bioavailable Amidobenzimidazole Analogues Targeting Stimulator of Interferon Gene (STING) Receptor.

Stimulator of interferon gene (STING) is a critical adaptor protein that has a pivotal role in triggering inherent immune responses to infection. STING-linked interferon production has been involved in anti-inflammation, anti-infection, and antitumor immunity. Herein, a series of amidobenzimidazole analogues as STING agonists were profiled for potency and drug-like properties. By structure-based modification and optimization based on mono-aminobenzimidazole (ABZI), analogues with nanomolar STING agonistic activities were obtained. Among them, compounds D59 and D61 significantly increased the transcription of IFN-ß and proinflammatory cytokine CXCL10, as well as dramatically induced the phosphorylation of STING downstream proteins in THP1 cells. Furthermore, compound D61 exhibited favorable pharmacokinetic properties and metabolic stabilities. In a CT-26 syngeneic mice-bearing tumor model, D61 effectively inhibited tumor growth with good tolerance when administered via intratumoral, intravenous, intraperitoneal, and oral routes. This research on orally bioavailable amidobenzimidazole analogues expands the diversity of chemical structures of agonists for STING-mediated immunotherapy.

Discovery of 2-((2-methylbenzyl)thio)-6-oxo-4-(3,4,5-trimethoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile as a novel and effective bromodomain and extra-terminal (BET) inhibitor for the treatment of sepsis.

Sepsis has long been a major health problem worldwide. It threatens the lives of hospitalized patients and has been one of the leading causes of death in hospitalized patients over the past decades. BRD4 has been regarded as a potential target for sepsis therapy, for its critical role in the transcriptional expression of NF-κB pathway-dependent inflammatory factors. In this study, compound 1 was obtained through virtual screening, and candidate compound 27 was obtained through several rounds of iterative SAR analysis. 27 decreased LPS-induced NO production and expression of the pro-inflammatory factors IL-6, IL-1ß and TNF-α. In vivo, 27 effectively protected mice from LPS-induced sepsis, increased survival rate and decreased the level of pro-inflammatory factors in serum. Collectively, we reported here 27, a BRD4 inhibitor with a new scaffold, as a potential candidate for the treatment of sepsis.

Target Fishing Reveals a Novel Mechanism of 1,2,4-Oxadiazole Derivatives Targeting Rpn6, a Subunit of 26S Proteasome.

1,2,4-Oxadiazole derivatives, a class of Nrf2-ARE activators, exert an extensive therapeutic effect on inflammation, cancer, neurodegeneration, and microbial infection. Among these analogues, DDO-7263 is the most potent Nrf2 activator and used as the core structure for bioactive probes to explore the precise mechanism. In this work, we obtained compound 7, a mimic of DDO-7263, and biotin-labeled and fluorescein-based probes, which exhibited homologous biological activities to DDO-7263, including activating Nrf2 and its downstream target genes, anti-oxidative stress, and anti-inflammatory effects. Affinity chromatography and mass analysis techniques revealed Rpn6 as the potential target protein regulating the Nrf2 signaling pathway. In vitro affinity experiments further confirmed that DDO-7263 upregulated Nrf2 through binding to Rpn6 to block the assembly of 26S proteasome and the subsequent degradation of ubiquitinated Nrf2. These results indicated that Rpn6 is a promising candidate target to activate the Nrf2 pathway for protecting cells and tissues from oxidative, electrophilic, and exogenous microbial stimulation.

Regulation of Nrf2 by phosphorylation: Consequences for biological function and therapeutic implications.

The transcription factor nuclear factor erythroid-derived 2-like 2 (NRF2) participates in the activation of the antioxidant cytoprotective pathway and other important physiological processes to maintain cellular homeostasis. The dysregulation of NRF2 activity plays a role in various diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. Thus, NRF2 activity is tightly regulated through multiple mechanisms, among which phosphorylation by kinases is critical in the posttranslational regulation of NRF2. For instance, PKC, casein kinase 2, and AMP-activated kinase positively, while GSK-3 negatively regulates NRF2 activity through phosphorylation of different sites. Here, we provide an overview of the phosphorylation regulation pattern of NRF2 and discuss the therapeutic potential of interventions targeting NRF2 phosphorylation.

Influence of sugammadex versus neostigmine for neuromuscular block reversal on the incidence of postoperative pulmonary complications: a meta-analysis of randomized controlled trials.

BACKGROUND: The influence of sugammadex for reversal of neuromuscular block (NMB) on postoperative pulmonary complications (PPCs), compared with neostigmine, remains to be determined. We performed a meta-analysis of randomized controlled trials (RCTs) to compare the incidence of PPCs between patients who received sugammadex versus neostigmine. METHODS: Relevant studies were obtained by searching the PubMed, Embase, and Cochrane Library databases. A random effects model incorporating the potential heterogeneity was used to pool the results. RESULTS: Fourteen RCTs including 1478 adult patients who underwent surgeries with general anesthesia were included, and of these, 753 received sugammadex and 725 received neostigmine for reversal of NMB. The pooled results showed that sugammadex was associated with a lower risk of overall PPCs compared to neostigmine (odds ratio [OR]: 0.62, 95% confidence interval [CI]: 0.43-0.89, p = 0.01; I2 = 0%). This finding remained consistent after exclusion of two studies with potential overlapping events (OR: 0.58, 95% CI: 0.36-0.96, p = 0.03; I2=9%). Stratified analyses according to the categories of PPCs showed that sugammadex was associated with a significantly lower risk of postoperative respiratory failure (OR: 0.60, 95% CI: 0.38-0.97, p = 0.04; I2 = 0%) but not of postoperative pulmonary infection (OR: 0.79, p = 0.71), atelectasis (OR: 0.78, p = 0.33), or pneumothorax (OR: 0.87, p = 0.79). CONCLUSIONS: Compared with neostigmine, the use of sugammadex for reversal of NMB was associated with a lower risk of PPCs, mainly due to a lower incidence of postoperative respiratory failure with the use of sugammadex.

Discovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability.

Myeloid cell leukemia 1 (Mcl-1) protein is a key negative regulator of apoptosis, and developing Mcl-1 inhibitors has been an attractive strategy for cancer therapy. Herein, we describe the rational design, synthesis, and structure-activity relationship study of 3,5-dimethyl-4-sulfonyl-1H-pyrrole-based compounds as Mcl-1 inhibitors. Stepwise optimizations of hit compound 11 with primary Mcl-1 inhibition (52%@30 µM) led to the discovery of the most potent compound 40 with high affinity (Kd = 0.23 nM) and superior selectivity over other Bcl-2 family proteins (>40,000 folds). Mechanistic studies revealed that 40 could activate the apoptosis signal pathway in an Mcl-1-dependent manner. 40 exhibited favorable physicochemical properties and pharmacokinetic profiles (F% = 41.3%). Furthermore, oral administration of 40 was well tolerated to effectively inhibit tumor growth (T/C = 37.3%) in MV4-11 xenograft models. Collectively, these findings implicate that compound 40 is a promising antitumor agent that deserves further preclinical evaluations.

Myeloid cell leukemin-1 inhibitors: a growing arsenal for cancer therapy.

B-cell lymphoma-2 (Bcl-2) family proteins, comprising proapoptotic proteins (Bax and Bak), antiapoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1) and BCL-2 homology domain 3 (BH3)-only proteins (Bid, Noxa, and Puma), have long been identified as pivotal apoptosis regulators. As an antiapoptotic member, myeloid cell leukemin-1 (Mcl-1) can bind with proapoptotic proteins and inhibit apoptosis. Mcl-1 is frequently overexpressed and closely associated with oncogenesis and poor prognosis in several cancers, posing a tremendous obstacle for cancer therapy. Recently, an increasing number of Mcl-1-selective small-molecule inhibitors have entered preclinical studies and advanced into clinical trials. In this review, we briefly introduce the role of Mcl-1 in apoptosis and highlight the recent development of Mcl-1 small-molecule inhibitors.

Discovery of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 protein-protein interaction inhibitors for inflammatory conditions.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a pleiotropic transcription factor which regulates the constitutive and inducible transcription of a wide array of genes and confers protection against a variety of pathologies. Directly disrupting Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 protein-protein interaction (PPI) has been explored as a promising strategy to activate NRF2. We reported here the first identification of a series of 2-oxy-2-phenylacetic acid substituted naphthalene sulfonamide derivatives as potent KEAP1-NRF2 inhibitors. Our efforts led to the potent small molecule KEAP1-NRF2 inhibitor, 20c, which exhibited a Kd of 24 nM to KEAP1 and an IC50 of 75 nM in disrupting KEAP1-NRF2 interaction. Subsequent biological studies provided consistent evidence across mouse macrophage cell-based and in vivo models that 20c induced NRF2 target gene expression and enhanced downstream antioxidant and anti-inflammatory activities. Our study not only demonstrated that small molecule KEAP1-NRF2 PPI inhibitors can be potential preventive and therapeutic agents for diseases and conditions involving oxidative stress and inflammation but also enriched the chemical diversity of the KEAP1-NRF2 inhibitors.

Azo-PROTAC: Novel Light-Controlled Small-Molecule Tool for Protein Knockdown.

Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable azobenzene-proteolysis targeting chimeras (Azo-PROTACs) by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photoswitchability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off properties.

Recent progress in Keap1-Nrf2 protein-protein interaction inhibitors.

Therapeutic targeting the protein-protein interaction (PPI) of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its main regulator, Kelch-like ECH-Associating protein 1 (Keap1) has been emerged as a feasible way to combat oxidative stress related diseases, due to the key role of Nrf2 in oxidative stress regulation. In recent years, many efforts have been made to develop potent Keap1-Nrf2 inhibitors with new chemical structures. Various molecules with diverse chemical structures have been reported and some compounds exhibit high potency. This review summarizes peptide and small molecule Keap1-Nrf2 inhibitors reported recently. We also highlight the pharmacological effects and discuss the possible therapeutic application of Keap1-Nrf2 inhibitors.

Proton pump inhibitors selectively suppress MLL rearranged leukemia cells via disrupting MLL1-WDR5 protein-protein interaction.

Genetic rearrangements of the mixed lineage leukemia (MLL) leading to oncogenic MLL-fusion proteins (MLL-FPs). MLL-FPs occur in about 10% of acute leukemias and are associated with dismal prognosis and treatment outcomes which emphasized the need for new therapeutic strategies. In present study, by a cell-based screening in-house compound collection, we disclosed that Rabeprazole specially inhibited the proliferation of leukemia cells harboring MLL-FPs with little toxicity to non-MLL cells. Mechanism study showed Rabeprazole down-regulated the transcription of MLL-FPs related Hox and Meis1 genes and effectively inhibited MLL1 H3K4 methyltransferase (HMT) activity in MV4-11 cells bearing MLL-AF4 fusion protein. Displacement of MLL1 probe from WDR5 protein suggested that Rabeprazole may inhibit MLL1 HMT activity through disturbing MLL1-WDR5 protein-protein interaction. Moreover, other proton pump inhibitors (PPIs) also indicated the inhibition activity of MLL1-WDR5. Preliminary SARs showed the structural characteristics of PPIs were also essential for the activities of MLL1-WDR5 inhibition. Our results indicated the drug reposition of PPIs for MLL-rearranged leukemias and provided new insight for further optimization of targeting MLL1 methyltransferase activity, the MLL1-WDR5 interaction or WDR5.

Design, Synthesis, and Structure-Activity Relationships of Indoline-Based Kelch-like ECH-Associated Protein 1-Nuclear Factor (Erythroid-Derived 2)-Like 2 (Keap1-Nrf2) Protein-Protein Interaction Inhibitors.

The Keap1 (Kelch-like ECH-associated protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE (antioxidant response element) pathway is the major defending mechanism against oxidative stresses, and directly disrupting the Keap1-Nrf2 protein-protein interaction (PPI) has been an attractive strategy to target oxidative stress-related diseases, including cardiovascular diseases. Here, we describe the design, synthesis, and structure-activity relationships (SARs) of indoline-based compounds as potent Keap1-Nrf2 PPI inhibitors. Comprehensive SAR analysis and thermodynamics-guided optimization identified 19a as the most potent inhibitor in this series, with an IC50 of 22 nM in a competitive fluorescence polarization assay. Further evaluation indicated the proper drug-like properties of 19a. Compound 19a dose-dependently upregulated genes and protein level of Nrf2 as well as its downstream markers and showed protective effects against lipopolysaccharide-induced injury in both H9c2 cardiac cells and mouse models. Collectively, we reported here a novel indoline-based Keap1-Nrf2 PPI inhibitor as a potential cardioprotective agent.