Recent advances in the design of small molecular drugs with acrylamides covalent warheads.

In the development of covalent inhibitors, acrylamides warhead is one of the most popular classes of covalent warheads. In recent years, researchers have made different structural modifications to acrylamides warheads, resulting in the creation of fluorinated acrylamide warheads and cyano acrylamide warheads. These new warheads exhibit superior selectivity, intracellular accumulation, and pharmacokinetic properties. Additionally, although ketoamide warheads have been applied in the design of covalent inhibitors for viral proteins, it has not received sufficient attention. Combined with the studies in kinase inhibitors and antiviral drugs, this review presents the structural features and the progression of acrylamides warheads, offering a perspective on future research and development in this field.

Casitas b cell lymphoma­B (Cbl-b): A new therapeutic avenue for small-molecule immunotherapy.

Immunotherapy has revolutionized the area of cancer treatment. Although most immunotherapies now are antibodies targeting membrane checkpoint molecules, there is an increasing demand for small-molecule drugs that address intracellular pathways. The E3 ubiquitin ligase Casitas B cell lymphoma­b (Cbl-b) has been regarded as a promising intracellular immunotherapy target. Cbl-b regulates the downstream proteins of multiple membrane receptors and co-receptors, restricting the activation of the innate and adaptive immune system. Recently, Cbl-b inhibitors have been reported with promising effects on immune surveillance activation and anti-tumor efficacy. Several molecules have entered phase Ⅰ clinical trials. In this review, the biological rationale of Cbl-b as a promising target for cancer immunotherapy and the latest research progress of Cbl-b are summarized, with special emphasis on the allosteric small-molecule inhibitors of Cbl-b.

Ligation to Scavenging Strategy Enables On-Demand Termination of Targeted Protein Degradation.

Event-driven bifunctional molecules, typified by proteolysis targeting chimera (PROTAC) technology, have been successfully applied in degrading many proteins of interest (POI). Due to the unique catalytic mechanism, PROTACs will induce multiple cycles of degradation until the elimination of the target protein. Here, we propose a versatile "Ligation to scavenging" approach to terminate event-driven degradation for the first time. Ligation to the scavenging system consists of a TCO-modified dendrimer (PAMAM-G5-TCO) and tetrazine-modified PROTACs (Tz-PROTACs). PAMAM-G5-TCO can rapidly scavenge intracellular free PROTACs via an inverse electron demand Diels-Alder reaction and terminate the degradation of certain proteins in living cells. Thus, this work proposes a flexible chemical knockdown approach to adjust the levels of POI on-demand in living cells, which paves the way for controlled target protein degradation.

Protein Phosphatase 5-Recruiting Chimeras for Accelerating Apoptosis-Signal-Regulated Kinase 1 Dephosphorylation with Antiproliferative Activity.

A normal phosphorylation state is essential for the function of proteins. Biased regulation frequently results in morbidity, especially for the hyperphosphorylation of oncoproteins. The hyperphosphorylation of ASK1 at Thr838 leads to a persistently high activity state, which accelerates the course of gastric cancer. Under normal conditions, PP5 specifically dephosphorylates p-ASK1T838 in cells, thereby weakening ASK1 to a low-basal activity state. However, in tumor types, PP5 shows low activity with a self-inhibition mechanism, making p-ASK1T838 remain at a high level. Thus, we aim to design phosphatase recruitment chimeras (PHORCs) through a proximity-mediated effect for specifically accelerating the dephosphorylation of p-ASK1T838. Herein, we describe DDO3711 as the first PP5-recruiting PHORC, which is formed by connecting a small molecular ASK1 inhibitor to a PP5 activator through a chemical linker, to effectively decrease the level of p-ASK1T838 in vitro and in vivo. DDO3711 shows preferable antiproliferative activity (IC50 = 0.5 µM) against MKN45 cells through a direct binding and proximity-mediated mechanism, while the ASK1 inhibitor and the PP5 activator, used alone or in combination, exhibit no effect on MKN45 cells. Using DDO3711, PHORCs are identified as effective tools to accelerate the dephosphorylation of POIs and provide important evidence to achieve precise phosphorylation regulation, which will promote confidence in the further regulation of abnormally phosphorylated oncoproteins.

Targeting m6A binding protein YTHDFs for cancer therapy.

N6-methyladenosine (m6A) is the most common mRNA modification in mammalians. The function and dynamic regulation of m6A depends on the "writer", "readers" and "erasers". YT521-B homology domain family (YTHDF) is a class of m6A binding proteins, including YTHDF1, YTHDF2 and YTHDF3. In recent years, the modification of m6A and the molecular mechanism of YTHDFs have been further understood. Growing evidence has shown that YTHDFs participate in multifarious bioprocesses, particularly tumorigenesis. In this review, we summarized the structural characteristics of YTHDFs, the regulation of mRNA by YTHDFs, the role of YTHDF proteins in human cancers and inhibition of YTHDFs.

Forward or Backward: Lessons Learned from Small Molecule Drugs Approved by FDA from 2012 to 2022.

At every juncture in history, the design and identification of new drugs pose significant challenges. To gain valuable insights for future drug development, we conducted a detailed analysis of New Molecular Entitiy (NME) approved by the Food and Drug Administration (FDA) from 2012 to 2022 and focused on the analysis of first-in-class (FIC) small-molecules from a perspective of a medicinal chemist. We compared the change of numbers between all the FDA-approved NMEs and FIC, which could be more visual to analyze the changing trend of FIC. To get a more visual change of molecular physical properties, we computed the annual average trends in molecular weight for FIC across various therapeutic fields. Furthermore, we consolidated essential information into three comprehensive databases, which covered the indications, canonical SMILES, structural formula, research and development (R&D) institutions, molecular weight, calculated LogP (CLogP), and route of administration on all the small-molecule pharmaceutical. Through the analysis of the database of 11 years of approvals, we forecast the development trend of NME approval in the future.

Targeting the HSP90-CDC37-kinase chaperone cycle: A promising therapeutic strategy for cancer.

Heat shock protein 90 (HSP90) is an indispensable molecular chaperone that facilitates the maturation of numerous oncoproteins in cancer cells, including protein kinases, ribonucleoproteins, steroid hormone receptors, and transcription factors. Although over 30 HSP90 inhibitors have steadily entered clinical trials, further clinical advancement has been restricted by their limited efficacy, inevitable heat shock response, and multiple side-effects, likely induced via an ATP inhibition mechanism. Since both ATP and various co-chaperones play essential roles in the HSP90 chaperone cycle to achieve integrated function, optimal therapeutics require an understanding of the dynamic interactions among HSP90, ATP, and cochaperones. To date, continuous research has promoted the exploration of the cochaperone cell division cycle 37 (CDC37) as a kinase-specific recognizer and has shown that the HSP90-CDC37-kinase complex is particularly relevant in cancers. Indeed, disrupting the HSP90-CDC37-kinase complex, rather than totally blocking the ATP function of HSP90, is emerging as an alternative way to avoid the limitations of current inhibitors. In this review, we first briefly introduce the HSP90-CDC37-kinase cycle and present the currently available approaches for inhibitor development targeting this cycle and provide insights into selective regulation of the kinase clients of HSP90 by more directional ways.

Recent applications of CBT-Cys click reaction in biological systems.

Click chemistry is a hot topic in many research fields. A biocompatible reaction from fireflies has attracted increasing attention since 2009. Herein, we focus on the firefly-sourced click reaction between cysteine (Cys) and 2-cyanobenzothiazole (2-CBT). This reaction has many excellent properties, such as rapidity, simplicity and high selectivity, which make it successfully applied in protein labeling, molecular imaging, drug discovery and other fields. Meanwhile, its unique ability to form nanoparticles expands its applications in biological systems. We review its principle, development, and latest applications in the past 5 years and hope this review provides more profound and comprehensive insights to its further application.

Design, synthesis, and biological evaluation of arylmethylpiperidines as Kv1.5 potassium channel inhibitors.

Kv1.5 potassium channel, encoded by KCNA5, is a promising target for the treatment of atrial fibrillation, one of the common arrhythmia. A new series of arylmethylpiperidines derivatives based on DDO-02001 were synthesised and evaluated for their ability to inhibit Kv1.5 channel. Among them, compound DDO-02005 showed good inhibitory activity (IC50 = 0.72 µM), preferable anti-arrhythmic effects and favoured safety. These results indicate that DDO-02005 can be a promising Kv1.5 inhibitor for further studies.

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 evaluation of phthalazinone thiohydantoin-based derivative as potent PARP-1 inhibitors.

Two new series of compounds were designed and synthesized as potent PARP-1 inhibitors. These compounds were evaluated for PARP-1 enzyme and cellular inhibitory activities. All efforts lead to the identification of 9k (named as LG-12) with efficient potency both for PARP-1 and BRCA1 deficient MDA-MB-436 cells. Additionally, the novel PARP-1 inhibitor LG-12 is an efficient chemosensitizer, which could potentiate the anti-cancer effect of TMZ. Our data presented herein provide a comprehensive preclinical in vitro evaluation of the potential therapeutic efficacy and potency of chemotherapeutic agent-PARP-1 inhibitor combinations for LG-12. The combined results indicated that LG-12 could be a promising candidate for further study.

Structure-guided design and synthesis of isoflavone analogs of GW4064 with potent lipid accumulation inhibitory activities.

Our group has previously reported a series of isoflavone derivatives with antidyslipidemic activity. With this background, a series of isoflavone analogs of GW4064 were designed, synthesized and evaluated the lipid-lowering activity of analogs. As a result, most of compounds significantly reduced the lipid accumulation in 3T3-L1 adipocytes and four of them (10a, 11, 15c and 15d) showed stronger inhibitory than GW4064. The most potent compound 15d exhibited promising agonistic activity for FXR in a cell-based luciferase reporter assay. Meanwhile, 15d up-regulated FXR, SHP and BSEP gene expression and down-regulated the mRNA expression of lipogenesis gene SREBP-1c. Besides, an improved safety profile of 15d was also observed in a HepG2 cytotoxicity assay compared with GW4064. The obtained biological results were further confirmed by a molecular docking study showing that 15d fitted well in the binding pocket of FXR and interacted with some key residues simultaneously.

Lipid accumulation inhibitory activities of novel isoxazole-based chenodeoxycholic acids: Design, synthesis and preliminary mechanism study.

In continuation of our drug discovery program on hyperlipidemia, a series of novel isoxazole-chenodeoxycholic acid hybrids were designed, synthesized and evaluated for their lipid-lowering effects. Preliminary screening of all the synthesized compounds was done by using a 3T3-L1 adipocyte model, in which the most active compound 16b could significantly reduce the lipid accumulation up to 30.5% at a nontoxic concentration 10 µM. Further mechanism studies revealed that 16b blocked lipid accumulation via activating FXR-SHP signaling pathway, efficiently down-regulated the expression of key lipogenesis regulator SREBP-1c.

Synthesis and biological evaluation of 4,6-diaryl-2-pyrimidinamine derivatives as anti-breast cancer agents.

Breast cancer is the most frequently diagnosed cancers and the leading causes of cancer death among females worldwide. Estrogen receptor positive has been identified as the predominant internal reasons, involving in more than 70% breast cancer patients and SERMs which competes with estradiol for the binding to ERα in breast tissue are widely used in the treatment of ER+ breast cancer, such as tamoxifen, raloxifene. However, many SERMs may cause negative side effects due to their estrogenic activity in other tissues and approximate 50% of patients with ER-positive tumors either initially do not respond or become resistant to these drugs. Here, a series of designed 4,6-diaryl-2-pyrimidinamine derivatives had been synthesized to treat estrogen receptor positive breast cancer by simultaneously antagonizing ER and inhibiting VEGFR-2. Bioactivity evaluation showed that these compounds could significantly inhibit the proliferation of MCF-7, HUVEC and Ishikawa cells. Further studies identified compound III-3A could antagonize against estrogen action and inhibit the phosphorylation of VEGFR-2 as well as inhibit angiogenesis in vivo. The results indicated designed 4,6-diaryl-2-pyrimidinamine derivatives can be used to further study as anti-breast cancer drugs.

Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed.

Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-leukemia agents by inhibiting Btk and PI3Kδ.

Btk inhibitors and PI3Kδ inhibitors play crucial roles in the treatment of leukemia, and studies confirmed that the synergetic inhibition against Btk and PI3Kδ could gain an optimal response. Herein, a series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were designed and synthesized as dual Btk/PI3Kδ kinases inhibitors for the treatment of leukemia. Studies indicated that most compounds could suppress the proliferation of multiple leukemia or lymphoma cells (Raji, HL60 and K562 cells) at low micromolar concentrations in vitro. Further kinase assays identified several compounds could simultaneously inhibit Btk kinase and PI3Kδ kinase. Thereinto, compound 16b exhibited the best inhibitory activity (Btk: IC50 = 139 nM; PI3Kδ: IC50 = 275 nM) and showed some selectivity against PI3Kδ compared to PI3Kß/γ. Finally, the SAR of target compounds was preliminarily discussed combined with docking results. In brief, 16b possessed of the potency for the further optimization as anti-leukemia drugs by inhibiting simultaneously Btk kinase and PI3Kδ kinase.

Lipid reducing activity of novel cholic acid (CA) analogs: Design, synthesis and preliminary mechanism study.

Bile acids, initially discovered as endogenous ligands of farnesoid X receptor (FXR), play a central role in the regulation of triglyceride and cholesterol metabolism and have recently emerged as a privileged structure for interacting with nuclear receptors relevant to a large array of metabolic processes. In this paper, phenoxy containing cholic acid derivatives with excellent drug-likeness have been designed, synthesized, and assayed as agents against cholesterol accumulation in Raw264.7 macrophages. The most active compound 14b reduced total cholesterol accumulation in Raw264.7 cells up to 30.5% at non-toxic 10 µM and dosage-dependently attenuated oxLDL-induced foam cell formation. Western blotting and qPCR results demonstrate that 14b reduced both cholesterol and lipid in Raw264.7 cells through (1) increasing the expression of cholesterol transporters ABCA1 and ABCG1, (2) accelerating ApoA1-mediated cholesterol efflux. Through a cell-based luciferase reporter assay and molecular docking analysis, LXR was identified as the potential target for 14b. Interestingly, unlike conventional LXR agonist, 14b did not increase lipogenesis gene SREBP-1c expression. Overall, these diverse properties disclosed herein highlight the potential of 14b as a promising lead for further development of multifunctional agents in the therapy of cardiovascular disease.

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.

Design, synthesis and biological evaluation of novel 3-oxo-4-oxa-5α-androst-17ß-amide derivatives as dual 5α-reductase inhibitors and androgen receptor antagonists.

Prostate cancer (PCa) is the second leading cause of death in men. Recently, some researches have showed that 5α-reductase inhibitors were beneficial in PCa treatment as well. In this study, a series of novel 3-oxo-4-oxa-5α-androst-17ß-amide derivatives have been designed and synthesized in a more simple and convenient method. Most of the synthesized compounds displayed good 5α-reductase inhibitory activities and androgen receptor binding affinities. Their anti-proliferation activities in PC-3 and LNCaP cell lines were also evaluated and the results indicated that most of the synthesized compounds exhibited potent anti-proliferative activities. It is obvious that the androgen-dependent cell line LNCaP was much more sensitive than the androgen-independent cell line PC-3. Among all the synthesized compounds, 11d and 11k displayed the best inhibition activity with 4-fold more sensitive toward LNCaP than PC-3, which was consistent with their high affinities observed in AR binding assay. Molecular modeling studies suggested that 11k could bind to AR in a manner similar to the binding of dihydrotestosterone to AR. Compared to the finasteride, 11k showed a longer plasma half-life (4h) and a better bioavailability. Overall, based on biological activities data, compound 11d and 11k can be identified as potential dual 5α-reductase inhibitors and AR antagonists which might be of therapeutic importance for prostate cancer treatment.

Design, synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents.

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC50 value of 4.52±2.47µM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure-activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.