STATs, promising targets for the treatment of autoimmune and inflammatory diseases.

Cytokines play a crucial role in the pathophysiology of autoimmune and inflammatory diseases, with over 50 cytokines undergoing signal transduction through the Signal Transducers and Activators of Transcription (STAT) signaling pathway. Recent studies have solidly confirmed the pivotal role of STATs in autoimmune and inflammatory diseases. Therefore, this review provides a detailed summary of the immunological functions of STATs, focusing on exploring their mechanisms in various autoimmune and inflammatory diseases. Additionally, with the rapid advancement of structural biology in the field of drug discovery, many STAT inhibitors have been identified using structure-based drug design strategies. In this review, we also examine the structures of STAT proteins and compile the latest research on STAT inhibitors currently being tested in animal models and clinical trials for the treatment of immunological diseases, which emphasizes the feasibility of STATs as promising therapeutic targets and provides insights into the design of the next generation of STAT inhibitors.

Advancing Tumor-Targeted Chemo-Immunotherapy: Development of the CAR-M-derived Exosome-Drug Conjugate.

Traditional antibody-drug conjugates (ADCs) mainly suppress tumor growth through either chemotherapy with cytotoxic payloads or immunotherapy with immuno-modulators. However, a single therapeutic modality may limit their exploration. Herein, we developed a new type of drug conjugate termed CAR-EDC (CAR-M-derived exosome-drug conjugate) by using CAR-exosomes from CAR-M cells as the targeting drug carrier that contains a high level of CXCL10. CAR-exosomes could significantly enhance the immunological activation and migratory capacity of T lymphocytes and promote their differentiation into CD8+ T cells. It also increased the proportion of M1 macrophages. The CAR-EDC, covalently loaded with SN-38, was internalized into Raji cells through endocytosis mediated by the CAR molecules. It exerted excellent antitumor activity in vivo by virtue of not only chemotherapy by SN38 but also immunotherapy by CXCL10-mediated antitumor immunity. Generally, this study provides an exosome-drug conjugate system with enhanced antitumor effects over traditional ADCs through the synergism of chemotherapy and immunotherapy.

Discovery and Optimization of Novel SaFabI Inhibitors as Specific Therapeutic Agents for MRSA Infection.

As the rate-limiting enzyme in fatty acid biosynthesis, Staphylococcus aureus enoyl-acyl carrier protein reductase (SaFabI) emerges as a compelling target for combating methicillin-resistant S. aureus (MRSA) infections. Herein, compound 1, featuring a 4-(1H-benzo[d]imidazol-2-yl)pyrrolidin-2-one scaffold, was identified as a potent SaFabI inhibitor (IC50 = 976.8 nM) from an in-house library. Subsequent optimization yielded compound n31, with improved inhibitory efficacy on enzymatic activity (IC50 = 174.2 nM) and selective potency against S. aureus (MIC = 1-2 µg/mL). Mechanistically, n31 directly inhibited SaFabI in cellular contexts. Moreover, n31 exhibited favorable safety and pharmacokinetic profiles, and dose-dependently treated MRSA-induced skin infections, outperforming the approved drug, linezolid. The chiral separation of n31 resulted in (S)-n31, with superior activities (IC50 = 94.0 nM, MIC = 0.25-1 µg/mL) and in vivo therapeutic efficacy. In brief, our research proposes (S)-n31 as a promising candidate for SaFabI-targeted therapy, offering specific anti-S. aureus efficacy and potential for further development.

Discovery of 5-(Piperidin-4-yl)-1,2,4-oxadiazole Derivatives as a New Class of Human Caseinolytic Protease P Agonists for the Treatment of Hepatocellular Carcinoma.

Chemical agonism of human caseinolytic protease P (HsClpP) is increasingly being recognized as a potential anticancer strategy due to its critical role in maintaining mitochondrial homeostasis. We unveil the discovery of 5-(piperidin-4-yl)-1,2,4-oxadiazole derivatives as a novel class of HsClpP agonists and demonstrate for the first time the application of HsClpP agonists in the treatment of hepatocellular carcinoma (HCC) (Pace, A.; Pierro, P. The new era of 1,2,4-oxadiazoles. Org. Biomol. Chem. 2009, 7 (21), 4337-4348). Compound SL44 exhibited potent HsClpP agonistic activity in the α-casein hydrolysis assay (EC50 = 1.30 µM) and inhibited the proliferation of HCCLM3 cells (IC50 = 3.1 µM, 21.4-fold higher than hit ADX-47273). Mechanistically, SL44 induces degradation of respiratory chain complex subunits and leads to apoptosis in HCC cells. In vivo results demonstrated that SL44 has potent tumor growth inhibitory activity and has a superior safety profile compared to the kinase inhibitor sorafenib. Overall, we developed a novel class of HsClpP agonists that can potentially be used for the treatment of HCC.

Exploration of novel isoxazole-fused quinone derivatives as anti-colorectal cancer agents through inhibiting STAT3 and elevating ROS level.

Colorectal cancer (CRC) is trending to be a major health problem throughout the world. Therapeutics with dual modes of action have shown latent capacity to create ideal anti-tumor activity. Signal transducer and activator of transcription 3 (STAT3) has been proved to be a potential target for the development of anti-colon cancer drug. In addition, modulation of tumor redox homeostasis through deploying exogenous reactive oxygen species (ROS)-enhancing agents has been widely applied as anti-tumor strategy. Thus, simultaneously targeting STAT3 and modulation ROS balance would offer a fresh avenue to combat CRC. In this work, we designed and synthesized a novel series of isoxazole-fused quinones, which were evaluated for their preliminary anti-proliferative activity against HCT116 cells. Among these quinones, compound 41 exerted excellent in vitro anti-tumor effect against HCT116 cell line with an IC50 value of 10.18 ± 0.4 nM. Compound 41 was proved to bind to STAT3 by using Bio-Layer Interferometry (BLI) assay, and can significantly inhibit phosphorylation of STAT3. It also elevated ROS of HCT116 cells by acting as a substrate of NQO1. Mitochondrial dysfunction, apoptosis, and cell cycle arrest, which was caused by compound 41, might be partially due to the inhibition of STAT3 phosphorylation and ROS production induced by 41. Moreover, it exhibited ideal anti-tumor activity in human colorectal cancer xenograft model and good safety profiles in vivo. Overall, this study provided a novel quinone derivative 41 with excellent anti-tumor activity by inhibiting STAT3 and elevating ROS level, and gave insights into designing novel anti-tumor therapeutics by simultaneously modulation of STAT3 and ROS.

In vitro and in vivo enhancement effect of glabridin on the antibacterial activity of colistin, against multidrug resistant Escherichia coli strains.

BACKGROUND: The increase in antimicrobial resistance leads to complications in treatments, prolonged hospitalization, and increased mortality. Glabridin (GLA) is a hydroxyisoflavan from Glycyrrhiza glabra L. that exhibits multiple pharmacological activities. Colistin (COL), a last-resort antibiotic, is increasingly being used in clinic against Gram-negative bacteria. Previous reports have shown that GLA is able to sensitize first line antibiotics such as norfloxacin and vancomycin on Staphylococcus aureus, implying that the use of GLA as an antibiotic adjuvant is a promising strategy for addressing the issue of drug resistance. However, the adjuvant effect on other antibiotics, especially COL, on Gram-negative bacteria such as Escherichia coli has not been studied. PURPOSE: The objective of our study was to investigate the targets of GLA and the synergistic effect of GLA and COL in E. coli, and to provide further evidence for the use of GLA as an antibiotic adjuvant to alleviate the problem of drug resistance. METHODS: We first investigated the interaction between GLA and enoyl-acyl carrier protein reductase, also called "FabI", through enzyme inhibition assay, differential scanning fluorimetry, isothermal titration calorimetry and molecular docking assay. We tested the transmembrane capacity of GLA on its own and combined it with several antibiotics. The antimicrobial activities of GLA and COL were evaluated against six different susceptible and resistant E. coli in vitro. Their interactions were analyzed using checkerboard assay, time-kill curve and CompuSyn software. A series of sensitivity tests was conducted in E. coli overexpressing the fabI gene. The development of COL resistance in the presence of GLA was tested. The antimicrobial efficacy of GLA and COL in a mouse model of urinary tract infection was assessed. The anti-biofilm effects of GLA and COL were investigated. RESULTS: In this study, enzyme kinetic analysis and thermal analysis provided evidence for the interaction between GLA and FabI in E. coli. GLA enhanced the antimicrobial effect of COL and synergistically suppressed six different susceptible and resistant E. coli with COL. Overexpression experiments showed that targeted inhibition of FabI was a key mechanism by which GLA synergistically enhanced COL activity. The combination of GLA and COL slowed the development of COL resistance in E. coli. Combined GLA and COL treatment significantly reduced bacterial load and mitigated urinary tract injury in a mouse model of E. coli urinary tract infection. Additionally, GLA + COL inhibited the formation and eradication of biofilms and the synthesis of curli. CONCLUSION: Our findings indicate that GLA synergistically enhances antimicrobial activities of COL by targeting inhibition of FabI in E. coli. GLA is expected to continue to be developed as an antibiotic adjuvant to address drug resistance issues.

Rational Design of a Novel Class of Human ClpP Agonists through a Ring-Opening Strategy with Enhanced Antileukemia Activity.

The activation of Homo sapiens Casein lysing protease P (HsClpP) by a chemical or genetic strategy has been proved to be a new potential therapy in acute myeloid leukemia (AML). However, limited efficacy has been achieved with classic agonist imipridone ONC201. Here, a novel class of HsClpP agonists is designed and synthesized using a ring-opening strategy based on the lead compound 1 reported in our previous study. Among these novel scaffold agonists, compound 7k exhibited remarkably enhanced proteolytic activity of HsClpP (EC50 = 0.79 ± 0.03 µM) and antitumor activity in vitro (IC50 = 0.038 ± 0.003 µM). Moreover, the intraperitoneal administration of compound 7k markedly suppressed tumor growth in Mv4-11 xenograft models, achieving a tumor growth inhibition rate of 88%. Concurrently, 7k displayed advantageous pharmacokinetic properties in vivo. This study underscores the promise of compound 7k as a significant HsClpP agonist and an antileukemia drug candidate, warranting further exploration for AML treatment.

Application of covalent modality in proximity-induced drug pharmacology: Early development, current strategy, and feature directions.

With a growing number of covalent drugs securing FDA approval as successful therapies across various indications, particularly in the realm of cancer treatment, the covalent modulating strategy is undergoing a resurgence. The renewed interest in covalent bioactive compounds has captured significant attention from both the academic and biopharmaceutical industry sectors. Covalent chemistry presents several advantages over traditional noncovalent proximity-induced drugs, including heightened potency, reduced molecular size, and the ability to target "undruggable" entities. Within this perspective, we have compiled a comprehensive overview of current covalent modalities applied to proximity-induced molecules, delving into their advantages and drawbacks. Our aim is to stimulate more profound insights and ideas within the scientific community, guiding future research endeavors in this dynamic field.

Design, Synthesis, and Biological Evaluation of 5-(5-Iodo-2-isopropyl-4-methoxyphenoxy)pyrimidine-2,4-diamine (AF-353) Derivatives as Novel DHFR Inhibitors against Staphylococcus aureus.

The high lethality of Staphylococcus aureus infections and the emergence of antibiotic resistance make the development of new antibiotics urgent. Our previous work identified a hit compound h1 (AF-353) as a novel Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor. Herein, we analyzed the antimicrobial profile of h1 and performed a comprehensive structure-activity relationship (SAR) assay based on h1. The representative compound j9 exhibited potent antibacterial activity against S. aureus without cross-resistance to other antimicrobial classes. Multiple genetic and biochemical approaches showed that j9 directly binds to SaDHFR, resulting in strong inhibition of its enzymatic activity (IC50 = 0.97 nM). Additionally, j9 had an acceptable in vivo safety profile and oral bioavailability (F = 40.7%) and also showed favorable efficacy in a mouse model of methicillin-resistant S. aureus (MRSA) skin infection. Collectively, these findings identified j9 as a novel SaDHFR inhibitor with the potential to combat drug-resistant S. aureus infections.

Discovery of a Novel Series of Homo sapiens Caseinolytic Protease P Agonists for Colorectal Adenocarcinoma Treatment via ATF3-Dependent Integrated Stress Response.

Homo sapiens caseinolytic protease P (HsClpP) activation is a promising strategy for colon cancer treatment. In this study, CCG1423 was identified as a selective activator of HsClpP. After optimization, NCA029 emerged as the most potent compound, with an EC50 of 0.2 µM against HsClpP. Molecular dynamics revealed that the affinity of NCA029 for the YYW aromatic network is crucial for its selectivity toward HsClpP. Furthermore, NCA029 displayed favorable pharmacokinetics and safety profiles and significantly inhibited tumor growth in HCT116 xenografts, resulting in 83.6% tumor inhibition. Mechanistically, NCA029 targeted HsClpP, inducing mitochondrial dysfunction and activating the ATF3-dependent integrated stress response, ultimately causing cell death in colorectal adenocarcinoma. These findings highlight NCA029 as an effective HsClpP activator with potential for colon cancer therapy.

Repurposing TAK875 as a novel STAT3 inhibitor for treating inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic immune-mediated disease associated with a high recurrence rate and an elevated risk of colon cancer. In this study, we screened a bioactive compound library using a luciferase reporter assay and identified the compound TAK875 as a novel inhibitor of signal transducer and activator of transcription 3 (STAT3). Surface plasmon resonance analysis, differential scanning fluorimetry, and isothermal titration calorimetry demonstrated that TAK875 directly bound to recombinant STAT3. TAK875 suppressed the lipopolysaccharide (LPS)-induced release of nitric oxide, inducible nitric oxide synthase, and inflammatory factors in RAW264.7 cells, likely by inhibiting STAT3 phosphorylation. In addition, TAK875 inhibited the differentiation of CD4+ T cells into T-helper 17 cells, which may partially account for its anti-inflammatory effect. TAK875 also alleviated the LPS-induced accumulation of intracellular reactive oxygen species, thus displaying its antioxidant effects. Finally, we demonstrated its satisfactory anti-inflammatory effect in a dextran sulfate sodium-induced mouse model of ulcerative colitis. In conclusion, this study presented TAK875 as a novel STAT3 inhibitor and demonstrated its anti-inflammatory and antioxidant effects both in vitro and in vivo.

Discovery and Mechanistic Study of Novel Mycobacterium tuberculosis ClpP1P2 Inhibitors.

Caseinolytic protease P (ClpP) responsible for the proteolysis of damaged or misfolded proteins plays a critical role in proteome homeostasis. MtbClpP1P2, a ClpP enzyme complex, is required for survival in Mycobacterium tuberculosis, and it is therefore considered as a promising target for the development of antituberculosis drugs. Here, we discovered that cediranib and some of its derivatives are potent MtbClpP1P2 inhibitors and suppress M. tuberculosis growth. Protein pull-down and loss-of-function assays validated the in situ targeting of MtbClpP1P2 by cediranib and its active derivatives. Structural and mutational studies revealed that cediranib binds to MtbClpP1P2 by binding to an allosteric pocket at the equatorial handle domain of the MtbClpP1 subunit, which represents a unique binding mode compared to other known ClpP modulators. These findings provide us insights for rational drug design of antituberculosis therapies and implications for our understanding of the biological activity of MtbClpP1P2.

Pseudomonas aeruginosa regulator PvrA binds simultaneously to multiple pseudo-palindromic sites for efficient transcription activation.

Tetracycline repressor (TetR) family regulators (TFRs) are the largest group of DNA-binding transcription factors and are widely distributed in bacteria and archaea. TFRs play vital roles in controlling the expression of various genes and regulating diverse physiological processes. Recently, a TFR protein Pseudomonas virulence regulator A (PvrA), was identified from Pseudomonas aeruginosa as the transcriptional activator of genes involved in fatty acid utilization and bacterial virulence. Here, we show that PvrA can simultaneously bind to multiple pseudo-palindromic sites and upregulate the expression levels of target genes. Cryo-electron microscopy (cryo-EM) analysis indicates the simultaneous DNA recognition mechanism of PvrA and suggests that the bound DNA fragments consist of a distorted B-DNA double helix. The crystal structure and functional analysis of PvrA reveal a hinge region that secures the correct domain motion for recognition of the promiscuous promoter. Additionally, our results showed that mutations disrupting the regulatory hinge region have differential effects on biofilm formation and pyocyanin biosynthesis, resulting in attenuated bacterial virulence. Collectively, these findings will improve the understanding of the relationship between the structure and function of the TetR family and provide new insights into the mechanism of regulation of P. aeruginosa virulence.

Strategies for the Discovery of Oxazolidinone Antibacterial Agents: Development and Future Perspectives.

Oxazolidinones represent a significant class of synthetic bacterial protein synthesis inhibitors that are primarily effective against Gram-positive bacteria. The commercial success of linezolid, the first FDA-approved oxazolidinone antibiotic, has motivated researchers to develop more potent oxazolidinones by employing various drug development strategies to fight against antimicrobial resistance, some of which have shown promising results. Thus, this Perspective aims to discuss the strategies employed in constructing oxazolidinone-based antibacterial agents and summarize recent advances in discovering oxazolidinone antibiotics to provide valuable insights for potentially developing next-generation oxazolidinone antibacterial agents or other pharmaceuticals.

Discovery and Optimization of Novel hDHODH Inhibitors for the Treatment of Inflammatory Bowel Disease.

As a key rate-limiting enzyme in the de novo synthesis of pyrimidine nucleotides, human dihydroorotate dehydrogenase (hDHODH) is considered a known target for the treatment of autoimmune diseases, including inflammatory bowel disease (IBD). Herein, BAY 41-2272 with a 1H-pyrazolo[3,4-b]pyridine scaffold was identified as an hDHODH inhibitor by screening an active compound library containing 5091 molecules. Further optimization led to 2-(1-(2-chloro-6-fluorobenzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-cyclopropylpyrimidin-4-amine (w2), which was found to be the most promising and drug-like compound with potent inhibitory activity against hDHODH (IC50 = 173.4 nM). Compound w2 demonstrated acceptable pharmacokinetic characteristics and alleviated the severity of acute ulcerative colitis induced by dextran sulfate sodium in a dose-dependent manner. Notably, w2 exerted better therapeutic effects on ulcerative colitis than hDHODH inhibitor vidofludimus and Janus kinase (JAK) inhibitor tofacitinib. Taken together, w2 is a promising hDHODH inhibitor for the treatment of IBD and deserves to be developed as a preclinical candidate.

Mycobacterium tuberculosis: Pathogenesis and therapeutic targets.

Tuberculosis (TB) remains a significant public health concern in the 21st century, especially due to drug resistance, coinfection with diseases like immunodeficiency syndrome (AIDS) and coronavirus disease 2019, and the lengthy and costly treatment protocols. In this review, we summarize the pathogenesis of TB infection, therapeutic targets, and corresponding modulators, including first-line medications, current clinical trial drugs and molecules in preclinical assessment. Understanding the mechanisms of Mycobacterium tuberculosis (Mtb) infection and important biological targets can lead to innovative treatments. While most antitubercular agents target pathogen-related processes, host-directed therapy (HDT) modalities addressing immune defense, survival mechanisms, and immunopathology also hold promise. Mtb's adaptation to the human host involves manipulating host cellular mechanisms, and HDT aims to disrupt this manipulation to enhance treatment effectiveness. Our review provides valuable insights for future anti-TB drug development efforts.

Synthesis and biological evaluation of novel pyrazole amides as potent mitochondrial complex I inhibitors.

Targeting mitochondrial complex I (CI) is emerging as an attractive anticancer strategy, and CI inhibitor IACS-010759 has achieved breakthrough success. However, the narrow therapeutic index of IACS-010759 seriously hinders its further application. In this study, a series of novel pyrazole amides were designed and optimized based on IACS-010759, and their potential CI inhibitory effects were biologically evaluated. Among them, the maximum tolerated dose (MTD) values of SCAL-255 (compound 5q) and SCAL-266 (compound 6f) were 68 mg/kg, which was nearly 10 times that of IACS-010759 (6 mg/kg), showing good safety. In addition, SCAL-255 and SCAL-266 significantly inhibited the proliferation of HCT116 and KG-1 cells in vitro and exerted satisfactory inhibitory activity against KG-1 cells in vivo. These results suggested that the optimized compounds might serve as promising CI inhibitors against oxidative phosphorylation (OXPHOS)-dependent cancer, which merits further study.

Small molecule II-6s synergises with fluconazole against Candida albicans.

BACKGROUND: Candida albicans (C. albicans) is the most common opportunistic fungal species in the oral cavity. The emergence of drug resistance of C. albicans has necessitated the development of novel antifungal agents. OBJECTIVES: This study evaluated the antifungal activity of a previously developed antimicrobial small molecule, namely II-6s, and explored its potential synergism with fluconazole against C. albicans and the underlying mechanisms. METHODS: The antifungal effects of II-6s against C. albicans were evaluated with microdilution and time-killing assays. Synergism of II-6s with fluconazole was determined by calculating the fractional inhibitory concentration index (FICI). The effects of II-6s on efflux pump, mitochondrial function and energy metabolism were examined to investigate the underlying mechanism of synergism. The antifungal mechanism of II-6s against C. albicans was further explored with RNA-seq and validated with specific mutant strains. RESULTS: II-6s exhibited a fungicidal effect against C. albicans with a minimum fungicidal concentration of 31.25 µg/mL. II-6s also inhibited C. albicans biofilm with a sessile minimum inhibitory concentration at 500 µg/mL. More importantly, II-6s showed a synergistic effect with fluconazole against a fluconazole-resistant strain of C. albicans, which expressed elevated levels of CDR1 (FICI < 0.5). II-6s inhibited the efflux pump activity of C. albicans. Consistently, II-6s inhibited energy metabolism of C. albicans by reducing the mitochondrial membrane potential and ATP generation, and inhibited utilisation of the non-fermentable carbon source. II-6s also inhibited the mitogen-activated protein kinase signal pathway, particularly HOG1, which may explain its antifungal activity against C. albicans. CONCLUSION: The small molecule II-6s inhibits the growth of C. albicans by targeting HOG1. II-6s also synergises with fluconazole by inhibiting the drug efflux pump, representing a promising antifungal agent for the control of candidiasis.

Structure-Activity Relationship of Novel Pyrimidine Derivatives with Potent Inhibitory Activities against Mycobacterium tuberculosis.

Discovery of novel antitubercular drugs is an effective strategy against drug-resistant tuberculosis (TB). Our previous study has identified LPX-16j as a novel antitubercular compound. Herein, we perform a comprehensive structure-activity relationship (SAR) based on LPX-16j, indicating that the central pyrimidine ring moiety was crucial for the antitubercular activities of its derivatives, and replacing the naphthyl group with hydrophobic substitutes was well tolerated. The representative derivative 5a exhibited potent activity against H37Ra, H37Rv, and clinical drug-resistant TB with minimum inhibitory concentration (MIC) values of 0.5-1.0 µg/mL. Meanwhile, 5a showed an acceptable safety in vivo and displayed a favorable oral bioavailability with a value of 40.7%. The differential scanning fluorescence, isothermal titration calorimetry, and molecular docking assays indicated that PknB could be one of the targets of compound 5a. Overall, this study identified 5a as a novel promising lead compound with the potential to develop candidates for the treatment of drug-resistant TB.

Targeting caseinolytic protease P and its AAA1 chaperone for tuberculosis treatment.

Caseinolytic protease P with its AAA1 chaperone, known as Mycobacterium tuberculosis (Mtb)ClpP1P2 proteolytic machinery, maintains protein homeostasis in Mtb cells and is essential for bacterial survival. It is regarded as an important biological target with the potential to address the increasingly serious issue of multidrug-resistant (MDR) TB. Over the past 10 years, many MtbClpP1P2-targeted modulators have been identified and characterized, some of which have shown potent anti-TB activity. In this review, we describe current understanding of the substrates, structure and function of MtbClpP1P2, classify the modulators of this important protein machine into several categories based on their binding subunits or pockets, and discuss their binding details; Such information provides insights for use in candidate drug research and development of TB treatments by targeting MtbClpP1P2 proteolytic machinery.