A deep learning-driven discovery of berberine derivatives as novel antibacterial against multidrug-resistant Helicobacter pylori.

Helicobacter pylori (H. pylori) is currently recognized as the primary carcinogenic pathogen associated with gastric tumorigenesis, and its high prevalence and resistance make it difficult to tackle. A graph neural network-based deep learning model, employing different training sets of 13,638 molecules for pre-training and fine-tuning, was aided in predicting and exploring novel molecules against H. pylori. A positively predicted novel berberine derivative 8 with 3,13-disubstituted alkene exhibited a potency against all tested drug-susceptible and resistant H. pylori strains with minimum inhibitory concentrations (MICs) of 0.25-0.5 µg/mL. Pharmacokinetic studies demonstrated an ideal gastric retention of 8, with the stomach concentration significantly higher than its MIC at 24 h post dose. Oral administration of 8 and omeprazole (OPZ) showed a comparable gastric bacterial reduction (2.2-log reduction) to the triple-therapy, namely OPZ + amoxicillin (AMX) + clarithromycin (CLA) without obvious disturbance on the intestinal flora. A combination of OPZ, AMX, CLA, and 8 could further decrease the bacteria load (2.8-log reduction). More importantly, the mono-therapy of 8 exhibited comparable eradication to both triple-therapy (OPZ + AMX + CLA) and quadruple-therapy (OPZ + AMX + CLA + bismuth citrate) groups. SecA and BamD, playing a major role in outer membrane protein (OMP) transport and assembling, were identified and verified as the direct targets of 8 by employing the chemoproteomics technique. In summary, by targeting the relatively conserved OMPs transport and assembling system, 8 has the potential to be developed as a novel anti-H. pylori candidate, especially for the eradication of drug-resistant strains.

Carrimycin inhibits coronavirus replication by decreasing the efficiency of programmed -1 ribosomal frameshifting through directly binding to the RNA pseudoknot of viral frameshift-stimulatory element.

The pandemic of SARS-CoV-2 worldwide with successive emerging variants urgently calls for small-molecule oral drugs with broad-spectrum antiviral activity. Here, we show that carrimycin, a new macrolide antibiotic in the clinic and an antiviral candidate for SARS-CoV-2 in phase III trials, decreases the efficiency of programmed -1 ribosomal frameshifting of coronaviruses and thus impedes viral replication in a broad-spectrum fashion. Carrimycin binds directly to the coronaviral frameshift-stimulatory element (FSE) RNA pseudoknot, interrupting the viral protein translation switch from ORF1a to ORF1b and thereby reducing the level of the core components of the viral replication and transcription complexes. Combined carrimycin with known viral replicase inhibitors yielded a synergistic inhibitory effect on coronaviruses. Because the FSE mechanism is essential in all coronaviruses, carrimycin could be a new broad-spectrum antiviral drug for human coronaviruses by directly targeting the conserved coronaviral FSE RNA. This finding may open a new direction in antiviral drug discovery for coronavirus variants.

Synthesis, Structure and Properties of Polyester Polyureas via a Non-Isocyanate Route with Good Combined Properties.

Polyureas have been widely applied in many fields, such as coatings, fibers, foams and dielectric materials. Traditionally, polyureas are prepared from isocyanates, which are highly toxic and harmful to humans and the environment. Synthesis of polyureas via non-isocyanate routes is green, environmentally friendly and sustainable. However, the application of non-isocyanate polyureas is quite restrained due to their brittleness as the result of the lack of a soft segment in their molecular blocks. To address this issue, we have prepared polyester polyureas via an isocyanate-free route and introduced polyester-based soft segments to improve their toughness and endow high impact resistance to the polyureas. In this paper, the soft segments of polyureas were synthesized by the esterification and polycondensation of dodecanedioic acid and 1,4-butanediol. Hard segments of polyureas were synthesized by melt polycondensation of urea and 1,10-diaminodecane without a catalyst or high pressure. A series of polyester polyureas were synthesized by the polycondensation of the soft and hard segments. These synthesized polyester-type polyureas exhibit excellent mechanical and thermal properties. Therefore, they have high potential to substitute traditional polyureas.

Discovery and development of novel 10,12-disubstituted aloperine derivatives against HCoV-OC43 by targeting allosteric site of host TMPRSS2.

By inducing steric activation of the 10CH bond with a 12-acyl group to form a key imine oxime intermediate, 20 novel (10S)-10,12-disubstituted aloperine derivatives were successfully synthesized and assessed for their antiviral efficacy against HCoV-OC43. Of them, compound 3i exhibited the moderate activities against HCoV-OC43, as well as against the SARS-CoV-2 variant EG.5.1 with the comparable EC50 values of 4.7 and 4.1 µM. A mechanism study revealed that it inhibited the protease activity of host TMPRSS2 by binding to an allosteric site, rather than the known catalytic center, different from that of camostat. Also, the combination of compound 3i and molnupiravir, as an RdRp inhibitor, showed an additive antiviral effect against HCoV-OC43. The results provide a new binding mode and lead compound for targeting TMPRSS2, with an advantage in combating broad-spectrum coronavirus.

Erratum: Author correction to 'Discovery and identification of EIF2AK2 as a direct key target of berberine for anti-inflammatory effects' [Acta Pharmaceutica Sinica B 13 (2023) 2138-2151].

[This corrects the article DOI: 10.1016/j.apsb.2022.12.009.].

Design, synthesis and biological evaluation of biaryl amide derivatives against SARS-CoV-2 with dual-target mechanism.

The COVID-19 pandemic highlights the urgent need to develop effective small-molecule antivirals. Thirty-three novel biaryl amide derivatives were synthesized and evaluated for anti-coronaviral activity. Some significant SARs were uncovered and the intensive structure modifications led to the most active compounds 8b and 8h. The broad-spectrum anti-coronaviral effects of 8h were validated at RNA and protein levels. 8h inhibits coronavirus replication at multiple stages, from virus entry to virus dsRNA synthesis. The mechanism of action showed that 8h may simultaneously act on 3CLpro and TMPRSS2 to display anti-coronaviral effects. 8h combined with RdRp inhibitor showed synergistic inhibitory activity against coronavirus. This study confirmed that biaryl amide derivatives may be a new class of potential therapeutic agents against coronavirus with multiple target effect, worthy of further investigation.

Design, synthesis and triglyceride-lowering activity of tricyclic matrine derivatives for the intervention of non-alcoholic fatty liver disease.

Thirty new tricyclicmatrinic derivatives were successively synthesized and evaluated for their inhibitory activity on the accumulation of triglycerides (TG) in AML12 cells, using 12 N-m-trifluoromethylbenzenesulfonyl matrine (1) as the hit compound. Among the analogues, compound 7n possessing 11-trimethylbutylamine quaternary exerted the highest in vitro TG-lowering potency, as well as a good safety profile. 7n significantly attenuated the hepatic injury and steatosis, and ameliorated dyslipidemia and dysglycemia in the mice with non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet. Primary mechanism study revealed that upregulation of peroxisome proliferator-activated receptors α (PPARα)-carnitine palmitoyltransferase 1A (CPT1A) pathway mediated the efficacy of 7n. Our study provides powerful information for developing this kind of compound into a new class of anti-NAFLD candidates, and compound 7n is worthy of further investigation as an ideal lead compound.

Evaluation and mechanism study of Pien Tze Huang against EV-A71 infection.

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) infection, currently lacks specific preventive and therapeutic interventions. Here, we demonstrated that Pien Tze Huang (PZH) could dose-dependently inhibit EV-A71 replication at the cellular level, resulting in significant reductions in EV-A71 virus protein 1 (VP1) expression and viral yields in Vero and human rhabdomyosarcoma cells. More importantly, we confirmed that PZH could protect mice from EV-A71 infection for the first time, with Ribavirin serving as a positive control. PZH treatment reduced EV-A71 VP1 protein expression, viral yields in infected muscles, and improved muscle pathology. Additionally, we conducted a preliminary mechanism study using quantitative proteomics. The results suggested that the suppression of the PI3K/AKT/mTOR and NF-κB signaling pathways may contribute to the anti-EV-A71 activity of PZH. These findings provide strong evidence supporting the potential therapeutic application of PZH for EV-A71 infection management.

USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1.

The abnormal upregulation of programmed death ligand-1 (PD-L1) on tumor cells impedes T-cell mediated cytotoxicity through PD-1 engagement, and further exploring the mechanisms regulation of PD-L1 in cancers may enhance the clinical efficacy of PD-L1 blockade. Here, using single-guide RNAs (sgRNAs) screening system, we identify ubiquitin-specific processing protease 2 (USP2) as a novel regulator of PD-L1 stabilization for tumor immune evasion. USP2 directly interacts with and increases PD-L1 abundance in colorectal and prostate cancer cells. Our results show that Thr288, Arg292 and Asp293 at USP2 control its binding to PD-L1 through deconjugating the K48-linked polyubiquitination at lysine 270 of PD-L1. Depletion of USP2 causes endoplasmic reticulum (ER)-associated degradation of PD-L1, thus attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell-mediated killing. Meanwhile, USP2 ablation-induced PD-L1 clearance enhances antitumor immunity in mice via increasing CD8+ T cells infiltration and reducing immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), whereas PD-L1 overexpression reverses the tumor growth suppression by USP2 silencing. USP2-depletion combination with anti-PD-1 also exhibits a synergistic anti-tumor effect. Furthermore, analysis of clinical tissue samples indicates that USP2 is positively associated with PD-L1 expression in cancer. Collectively, our data reveal a crucial role of USP2 for controlling PD-L1 stabilization in tumor cells, and highlight USP2 as a potential therapeutic target for cancer immunotherapy.

Synthesis of peptidomimetics as antibiotic adjuvants for combination with aztreonam to combat MDR Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the multipledrug-resistant (MDR) Gram-negative pathogens with few drugs available for treatment. Antibiotic adjuvant approach provides an alternative and complementary strategy. In this study, the stereo-structure-activity relationship of monobactams against MDR Gram-negative organisms was extended. Meanwhile, a series of novel peptidemimetic derivatives as antibiotic adjuvants was synthesized and evaluated for their synergistic effects with aztreonam (AZT) against P. aeruginosa, using dipeptide PAßN as the lead. Among the analogues, compound 22j showed a significant synergistic effect against MDR P. aeruginosa in vitro and in vivo, presumably through the mechanism of affecting the permeability of outer membrane. Thus, we identified 22j as a novel peptidemimetic lead compound to potentiate the activity of AZT against MDR P. aeruginosa, which is worthy of further development as antibiotic adjuvant candidates.

Evolution and development of potent monobactam sulfonate candidate IMBZ18g as a dual inhibitor against MDR Gram-negative bacteria producing ESBLs.

A series of new monobactam sulfonates is continuously synthesized and evaluated for their antimicrobial efficacies against Gram-negative bacteria. Compound 33a (IMBZ18G) is highly effective in vitro and in vivo against clinically intractable multi-drug-resistant (MDR) Gram-negative strains, with a highly druglike nature. The checkerboard assay reveals its significant synergistic effect with ß-lactamase inhibitor avibactam, and the MIC values against MDR enterobacteria were reduced up to 4-512 folds. X-ray co-crystal and chemoproteomic assays indicate that the anti-MDR bacteria effect of 33a results from the dual inhibition of the common PBP3 and some class A and C ß-lactamases. Accordingly, preclinical studies of 33a alone and 33a‒avibactam combination as potential innovative candidates are actively going on, in the treatment of ß-lactamase-producing MDR Gram-negative bacterial infections.

Evolution and Discovery of Matrine Derivatives as a New Class of Anti-Hepatic Fibrosis Agents Targeting Ewing Sarcoma Breakpoint Region 1 (EWSR1).

A series of new tricyclic matrinane derivatives were continuously synthesized and evaluated for their inhibitory effects on genes and proteins related to hepatic fibrosis at the cellular level, including collagen type I α1 chain (COL1A1), α smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and matrix metalloprotein 2 (MMP-2). Among them, compound 6k exerted an appealing potency and significantly reduced liver injury and fibrosis in both bile duct ligation (BDL) rats and Mdr2 knockout mice. An activity-based protein profiling (ABPP) assay indicated that 6k might directly bind to Ewing sarcoma breakpoint region 1 (EWSR1) to inhibit its function and affect the expression of downstream liver fibrosis-related genes and thus regulate liver fibrosis. These results provided a potential novel target for the treatment of liver fibrosis and powerful information for the development of tricyclic matrinanes into promising anti-hepatic fibrosis agents.

Discovery and identification of EIF2AK2 as a direct key target of berberine for anti-inflammatory effects.

Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF-κB, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1ß, IL-6, IL-18 and TNF-α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders.

Discovery and development of palmatine analogues as anti-NASH agents by activating farnesoid X receptor (FXR).

Sixty-one palmatine (PMT) derivatives, of which twenty-eight were new, were synthesized and evaluated for their anti-fibrogenic activities via collagen type I α 1 (COL1A1)-promoter based luciferase model in LX-2 cells, taking 2,3,10-trimethoxy-9-p-isopropyloxyprotopalmatine bromide (1) as the lead. Among them, compound 3a exerted the highest potency with the IC50 value of 8.19 µmol/L and SI value of 8.59, and reduced the expressions of multiple fibrogenic biomarkers, including COL1A1, TGF-ß1, α-SMA and TIMP1 in a dose-dependent manner. In addition, it significantly reduced liver steatosis and inflammation, and especially attenuated the degree of liver fibrosis in choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD)-induced NASH mice model in vivo. Mechanism study indicated that it significantly ameliorated liver injury by activating farnesoid X receptor (FXR). BDL-induced fibrosis rats model further verified its liver-protective and anti-fibrosis activities. Therefore, PMT derivatives constituted a new family of non-steroidal FXR agonists as anti-NASH candidates, with the advantage of good safety profile, and are worthy for further investigation.

Discovery of new riminophenazine analogues as antimycobacterial agents against drug-resistant Mycobacterium tuberculosis.

Twenty-three new riminophenazine and pyrido[3,2-b]quinoxaline derivatives were prepared and examined for their antimycobacterial activities against Mycobacterium marinum and Mycobacterium tuberculosis H37Rv, taking clofazimine (1) as the lead. Structure-activity relationship (SAR) analysis revealed that the introduction of a heterocycle or diethylamine substituted benzene moiety on the N-5 atom might be beneficial for activity. The most potent compound 7m also displayed enhanced activity against wild-type as well as multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB clinical isolates, with the MICs ranging from 0.08 to 1.25 µg/mL, especially effective toward strain M20A507, resistant to 1. Further mechanism study indicated that its anti-TB activity was independent of cell membrane disruption, but related to NDH-2 reduction and the resulting high ROS production. Our study provides instructive guidance for the further development of clofazimine derivatives into promising antimicrobial agents against MDR and XDR TB.

A Non-Isocyanate Route to Poly(Ether Urethane): Synthesis and Effect of Chemical Structures of Hard Segment.

A series of non-isocyanate poly(ether urethane) (PEU) were prepared by an environmentally friendly route based on dimethyl carbonate, diols and a polyether. The effect of the chemical structure of polyurethane hard segments on the properties of this kind of PEU was systematically investigated in this work. Polyurethane hard segments with different structures were first prepared from hexamethylene di-carbamate (BHC) and different diols (butanediol, hexanediol, octanediol and decanediol). Subsequently, a series of non-isocyanate PEU were obtained by polycondensation of the polyurethane hard segments with the polyether soft segments (PTMG2000). The PEU were characterized by GPC, FT-IR, 1H NMR, DSC, WAXD, SAXS, AFM and tensile testing. The results show that the urea groups generated by the side reaction affect the degree of crystallization of hard segments by influencing the hydrogen bonding of the hard segments molecular chains. The degree of hard segment crystallization, in turn, affects the thermal and mechanical properties of the polymer. The urea group content is related to the carbon chain length of the diol used for the synthesis of hard segments. When butanediol is applied to synthesize hard segment, the hard segment of the resulting PEU is unable to crystallize. Therefore, the tensile strength and modulus of elasticity of butanediol-based PEU is lowest among three, though it possesses the highest urea group content. When longer octanediol or decanediol is applied to synthesize the hard segment, the hard segments in the resulting polyether-based polyurethane are crystallizable and the resulting PEU possesses higher tensile strength.

Palmatine Derivatives as Potential Antiplatelet Aggregation Agents via Protein Kinase G/Vasodilator-Stimulated Phosphoprotein and Phosphatidylinositol 3-Kinase/Akt Phosphorylation.

Sixty palmatine (PMT) derivatives were synthesized and evaluated for antiplatelet aggregation taking berberine as the lead, and the structure-activity relationship was first systematically described. Among them, compound 2v showed the best potency in reducing adenosine diphosphate (ADP)-induced platelet aggregation in a dose-dependent manner. It greatly suppressed ADP-induced platelet aggregation, activation, and Akt phosphorylation in vitro and ex vivo after oral administration to mice. It also effectively inhibited carrageenan-induced thrombus formation in the mouse tail and lung, as well as reduced the serum P-selectin level. Compound 2v might simultaneously bind to protein kinase G to improve vasodilator-stimulated phosphoprotein phosphorylation and bind to phosphatidylinositol 3-kinase to inhibit Akt phosphorylation, which synergically reduced platelet aggregation, thereby achieving antithrombotic efficacy. Therefore, PMT derivatives constituted a novel family of antiplatelet aggregation agents with the advantage of a good safety profile, worthy of further investigation.

Clofazimine derivatives as potent broad-spectrum antiviral agents with dual-target mechanism.

Thirty-two clofazimine derivatives, of which twenty-two were new, were synthesized and evaluated for their antiviral effects against both rabies virus and pseudo-typed SARS-CoV-2, taking clofazimine (1) as the lead. Among them, compound 15f bearing 4-methoxy-2-pyridyl at the N5-position showed superior or comparable antiviral activities to lead 1, with the EC50 values of 1.45 µM and 14.6 µM and the SI values of 223 and 6.1, respectively. Compound 15f inhibited rabies and SARS-CoV-2 by targeting G or S protein to block membrane fusion, as well as binding to L protein or nsp13 to inhibit intracellular biosynthesis respectively, and thus synergistically exerted a broad-spectrum antiviral effect. The results provided useful scientific data for the development of clofazimine derivatives into a new class of broad-spectrum antiviral candidates.

Structure-activity relationship and biological evaluation of 12 N-substituted aloperine derivatives as PD-L1 down-regulatory agents through proteasome pathway.

Twenty-nine 12 N-substituted aloperine derivatives were synthesized and screened for suppression on PD-L1 expression in H460 cells, as a continuation of our work. Systematic structural modifications led to the identification of compound 6b as the most active PD-L1 modulator. Compound 6b could significantly down-regulate both constitutive and inductive PD-L1 expression in NSCLC cells, and successively enhance the cytotoxicity of co-cultured T cells against tumor cells at the concentration of 20 µM. Also, it exhibited a moderate in vivo anticancer efficacy against Lewis tumor xenograft with a stable PK and safety profile. The mechanism study indicated that 6b mediated the degradation of PD-L1 through a proteasome pathway, rather than a lysosome route. These results provided the powerful information for cancer immunotherapy of aloperine derivatives with unique endocyclic skeleton by targeting PD-L1 to activate immune cells to kill cancer cells.

Discovery and evolution of 12N-substituted aloperine derivatives as anti-SARS-CoV-2 agents through targeting late entry stage.

So far, there is still no specific drug against COVID-19. Taking compound 1 with anti-EBOV activity as the lead, fifty-four 12N-substituted aloperine derivatives were synthesized and evaluated for the anti-SARS-CoV-2 activities using pseudotyped virus model. Among them, 8a exhibited the most potential effects against both pseudotyped and authentic SARS-CoV-2, as well as SARS-CoV and MERS-CoV, indicating a broad-spectrum anti-coronavirus profile. The mechanism study disclosed that 8a might block a late stage of viral entry, mainly via inhibiting host cathepsin B activity rather than directly targeting cathepsin B protein. Also, 8a could significantly reduce the release of multiple inflammatory cytokines in a time- and dose-dependent manner, such as IL-6, IL-1ß, IL-8 and MCP-1, the major contributors to cytokine storm. Therefore, 8a is a promising agent with the advantages of broad-spectrum anti-coronavirus and anti-cytokine effects, thus worthy of further investigation.