Sulfonamidyl derivatives of sigmacidin: Protein-protein interaction inhibitors targeting bacterial RNA polymerase and sigma factor interaction exhibiting antimicrobial activity against antibiotic-resistant bacteria.

RNA polymerase is an essential enzyme involved in bacterial transcription, playing a crucial role in RNA synthesis. However, it requires the association with sigma factors to initiate this process. In our previous work, we utilized a structure-based drug discovery approach to create benzoyl and benzyl benzoic acid compounds. These compounds were designed based on the amino acid residues within the key binding site of sigma factors, which are crucial for their interaction with RNA polymerase. By inhibiting bacterial transcription, these compounds exhibited notable antimicrobial activity, and we coined them as sigmacidins to highlight their resemblance to sigma factors and the benzoic acid structure. In this study, we further modified the compound scaffolds and developed a series of sulfonamidyl benzoic acid derivatives. These derivatives displayed potent antimicrobial activity, with minimum inhibitory concentrations (MICs) as low as 1 µg/mL, demonstrating their efficacy against bacteria. Furthermore, these compounds demonstrated low cytotoxicity, indicating their potential as safe antimicrobial agents. To ascertain their mechanism of action in interfering with bacterial transcription, we conducted biochemical and cellular assays. Overall, this study showcases the effectiveness of sulfonamidyl benzoic acid derivatives as antimicrobial agents by targeting protein-protein interactions involving RNA polymerase and sigma factors. Their strong antimicrobial activity and low cytotoxicity implicate their potential in combating antibiotic-resistant bacteria.

A ratiometric fluorescence probe for selective and sensitive detection of leucine aminopeptidase in lysosome.

We designed a novel ratiometric fluorescent probe P1-Leu with a donor-acceptor-donor fluorophore for the detection of leucine aminopeptidase in lysosomes. P1-Leu exhibits a lower detection limit than the ratiometric donor-π-acceptor probe, due to the low ratiometric background. Besides, P1-Leu has good lysosome-targeting ability and realizes the distinction of LAP levels in different cells.

QSAR, Docking, and Molecular Dynamics Simulation Studies of Sigmacidins as Antimicrobials against Streptococci.

Streptococci are a family of bacterial species significantly affecting human health. In addition, environmental Streptococci represent one of the major causes of diverse livestock diseases. Due to antimicrobial resistance, there is an urgent need for novel antimicrobial agent discovery against Streptococci. We discovered a class of benzoic acid derivatives named sigmacidins inhibiting the bacterial RNA polymerase-σ factor interaction and demonstrating excellent antimicrobial activity against Streptococci. In this work, a combinational computer approach was applied to gain insight into the structural basis and mechanism of action of sigmacidins as antimicrobials against Streptococcus pneumoniae. Both two- and three-dimensional quantitative structure-active relationships (2D and 3D QSAR) of sigmacidins displayed good predictive ability. Moreover, molecular docking and molecular dynamics simulation studies disclosed possible contacts between the inhibitors and the protein. The results obtained in this study provided understanding and new directions to the further optimizations of sigmacidins as novel antimicrobials.

Inhibition of Influenza Virus Replication by Oseltamivir Derivatives.

Characterized by the high morbidity and mortality and seasonal surge, the influenza virus (IV) remains a major public health challenge. Oseltamivir is commonly used as a first-line antiviral. As a neuraminidase inhibitor, it attenuates the penetration of viruses through the mucus on the respiratory tract and inhibits the release of virus progeny from infected cells. However, over the years, oseltamivir-resistant strains have been detected in the IV surveillance programs. Therefore, new antivirals that circumvent the resistant strains would be of great importance. In this study, two novel secondary amine derivatives of oseltamivir CUHK326 (6f) and CUHK392 (10i), which bear heteroaryl groups of M2-S31 proton channel inhibitors, were designed, synthesized and subjected to biological evaluation using plaque assay. Influenza A virus (A/Oklahoma/447/2008, H1N1), influenza B viruses (B/HongKong/CUHK33261/2012), an oseltamivir-resistant influenza A virus (A/HongKong/CUHK71923/2009, H1N1) and an oseltamivir-resistant influenza B virus (B/HongKong/CUHK33280/2012) were included in the antiviral effect assessment compared to oseltamivir carboxylate (OC). Both novel compounds significantly reduced the plaque size of seasonal IV A and B, and performed similarly to OC at their corresponding half-maximal inhibitory concentration (IC50). CUHK392 (10i) functioned more effectively than CUHK326 (6f). More importantly, these compounds showed an inhibitory effect on the oseltamivir-resistant strain under 10 nM with selective index (SI) of >200.

Ligand-Based Drug Design of Novel Antimicrobials against Staphylococcus aureus by Targeting Bacterial Transcription.

Staphylococcus aureus is a common human commensal pathogen that causes a wide range of infectious diseases. Due to the generation of antimicrobial resistance, the pathogen becomes resistant to more and more antibiotics, resulting in methicillin-resistant S. aureus (MRSA) and even multidrug-resistant S. aureus (MDRSA), namely 'superbugs'. This situation highlights the urgent need for novel antimicrobials. Bacterial transcription, which is responsible for bacterial RNA synthesis, is a valid but underutilized target for developing antimicrobials. Previously, we reported a novel class of antimicrobials, coined nusbiarylins, that inhibited bacterial transcription by interrupting the protein-protein interaction (PPI) between two transcription factors NusB and NusE. In this work, we developed a ligand-based workflow based on the chemical structures of nusbiarylins and their activity against S. aureus. The ligand-based models-including the pharmacophore model, 3D QSAR, AutoQSAR, and ADME/T calculation-were integrated and used in the following virtual screening of the ChemDiv PPI database. As a result, four compounds, including J098-0498, 1067-0401, M013-0558, and F186-026, were identified as potential antimicrobials against S. aureus, with predicted pMIC values ranging from 3.8 to 4.2. The docking study showed that these molecules bound to NusB tightly with the binding free energy ranging from -58 to -66 kcal/mol.

Design, Synthesis, Biological Evaluation and In Silico Studies of Pyrazole-Based NH2-Acyl Oseltamivir Analogues as Potent Neuraminidase Inhibitors.

Oseltamivir represents one of the most successful neuraminidase (NA) inhibitors in the current anti-influenza therapy. The 150-cavity of NA was identified as an additional binding pocket, and novel NA inhibitors have been designed to occupy the 150-cavity based on the structure information of oseltamivir carboxylate (OC) in complex with NA. In this study, a series of C-5-NH2-acyl derivatives of OC containing the pyrazole moiety were synthesized. Several derivatives exhibited substantial inhibitory activity against NA. Moreover, in silico ADME evaluation indicated that the derivatives were drug-like with higher oral absorption rates and greater cell permeability than OC. Additionally, molecular docking studies revealed that the derivatives interacted with both the NA enzyme active site and 150-cavity as expected. The results provided useful information for further structural optimization of OC.

Benzyl and benzoyl benzoic acid inhibitors of bacterial RNA polymerase-sigma factor interaction.

Transcription is an essential biological process in bacteria requiring a core enzyme, RNA polymerase (RNAP). Bacterial RNAP is catalytically active but requires sigma (σ) factors for transcription of natural DNA templates. σ factor binds to RNAP to form a holoenzyme which specifically recognizes a promoter, melts the DNA duplex, and commences RNA synthesis. Inhibiting the binding of σ to RNAP is expected to inhibit bacterial transcription and growth. We previously identified a triaryl hit compound that mimics σ at its major binding site of RNAP, thereby inhibiting the RNAP holoenzyme formation. In this study, we modified this scaffold to provide a series of benzyl and benzoyl benzoic acid derivatives possessing improved antimicrobial activity. A representative compound demonstrated excellent activity against Staphylococcus epidermidis with minimum inhibitory concentrations reduced to 0.5 µg/mL, matching that of vancomycin. The molecular mechanism of inhibition was confirmed using biochemical and cellular assays. Low cytotoxicity and metabolic stability of compounds demonstrated the potential for further studies.

Severe recurrent Streptobacillus moniliformis endocarditis in a pregnant woman, and review of the literature.

BACKGROUND: Rat bite fever is a systemic febrile illness caused by infection with the Gram-negative bacillus Streptobacillus moniliformis following a bite, scratch, or contact with excrement. Only 26 cases of native valve endocarditis have been reported to date. We could find no other reports of severe Streptobacillus endocarditis requiring valve replacement in a young, pregnant patient. CASE PRESENTATION: A pregnant patient sought care for right leg pain, fevers, left upper quadrant pain, generalized weakness, fatigue, and inability to bear weight on her right leg. She had a syncopal episode 9 months earlier, resulting in a mandibular fracture and internal fixation hardware. Her pregnancy was complicated by hyperemesis and weight loss. Her pets included a rescued wild bird, a cat, and four rats. Her parents rescued stray cats, and she recalled multiple cat bites and scratches since childhood. She denied injection drug use. Ultrasound indicated a right popliteal artery thrombus. Transesophageal echocardiogram revealed a 2 cm × 0.7 cm vegetation. Angiography demonstrated multiple splenic infarcts and bilateral renal infarcts. She underwent mitral valve repair. The mitral valve Gram stain demonstrated 2+ Gram-negative rods, rare Gram-positive rods, and moderate white blood cells. Propionibacterium spp. was isolated from the mitral valve tissue on Columbia agar incubated anaerobically. Anaerobic and aerobic cultures of the valve tissue on all other broths and agars remained negative at 14 days. Hematoxylin and eosin stains showed a fibro-inflammatory vegetation. Aggregates of rod-shaped bacteria were identified on Warthin Starry/Steiner stain. Bartonella titers were positive for B. henselae IgG 1:256, IgM < 1:20. Brown-Hopps Gram stain, AFB, and GMS stains for bacterial and fungal microorganisms were negative. Broad range bacterial PCR and sequencing of a segment of 16 s rRNA gene of the valve tissue matched to Streptobacillus sp. (genus level) and most closely related to Streptobacillus moniliformis. CONCLUSION: This case demonstrates diagnostic and therapeutic challenges associated with a relatively uncommon cause of endocarditis. The diagnosis of rat bite fever was delayed due to symptoms of a concomitant pregnancy. Other confounders included possible alternative sources or co-infections with another zoonosis from multiple pets, and an odontogenic source due to presence of exposed jaw hardware.

Discovery of Antibacterials That Inhibit Bacterial RNA Polymerase Interactions with Sigma Factors.

Formation of a bacterial RNA polymerase (RNAP) holoenzyme by a catalytic core RNAP and a sigma (σ) initiation factor is essential for bacterial viability. As the primary binding site for the housekeeping σ factors, the RNAP clamp helix domain represents an attractive target for novel antimicrobial agent discovery. Previously, we designed a pharmacophore model based on the essential amino acids of the clamp helix, such as R278, R281, and I291 (Escherichia coli numbering), and identified hit compounds with antimicrobial activity that interfered with the core-σ interactions. In this work, we rationally designed and synthesized a class of triaryl derivatives of one hit compound and succeeded in drastically improving the antimicrobial activity against Streptococcus pneumoniae, with the minimum inhibitory concentration reduced from 256 to 1 µg/mL. Additional characterization of antimicrobial activity, inhibition of transcription, in vitro pharmacological properties, and cytotoxicity of the optimized compounds demonstrated their potential for further development.

Novel N-Substituted oseltamivir derivatives as potent influenza neuraminidase inhibitors: Design, synthesis, biological evaluation, ADME prediction and molecular docking studies.

The discovery of novel potent neuraminidase (NA) inhibitors remains an attractive approach for treating infectious diseases caused by influenza. In this study, we describe the design and synthesis of novel N-substituted oseltamivir derivatives for probing the 150-cavity which is nascent to the activity site of NA. NA inhibitory studies showed that new derivatives demonstrated the inhibitory activity with IC50 values at nM level against NA of a clinical influenza virus strain. Moreover, the in silico ADME predictions showed that the selected compounds had comparable properties with oseltamivir carboxylate, which demonstrated the druggablity of these derivatives. Furthermore, molecular docking studies showed that the most potent compound 6f and 10i could adopt different modes of binding interaction with NA, which may provide novel solutions for treating oseltamivir-resistant influenza. Based on the research results, we consider that compounds 6f and 10i have the potential for further studies as novel antiviral agents.

First-In-Class Inhibitors Targeting the Interaction between Bacterial RNA Polymerase and Sigma Initiation Factor Affect the Viability and Toxin Release of Streptococcus pneumoniae.

Novel antimicrobial classes are in desperate need for clinical management of infections caused by increasingly prevalent multi-drug resistant pathogens. The protein-protein interaction between bacterial RNA polymerase (RNAP) and the housekeeping sigma initiation factor is essential to transcription and bacterial viability. It also presents a potential target for antimicrobial discovery, for which a hit compound (C3) was previously identified from a pharmacophore model-based in silico screen. In this study, the hit compound was experimentally assessed with some rationally designed derivatives for the antimicrobial activities, in particular against Streptococcus pneumoniae and other pathogens. One compound, C3-005, shows dramatically improved activity against pneumococci compared to C3. C3-005 also attenuates S. pneumoniae toxin production more strongly than existing classes of antibiotics tested. Here we demonstrate a newly validated antimicrobial agent to address an overlooked target in the hit-to-lead process, which may pave the way for further antimicrobial development.

Simple Method for Studying in Vitro Protein-Protein Interactions Based on Protein Complementation and Its Application in Drug Screening Targeting Bacterial Transcription.

Protein-protein interactions (PPIs) underpin essential cellular processes of all organisms and are increasingly considered as drug targets. A number of techniques have been established to study PPIs; however, development of a simple and cost-effective method for in vitro high throughput screening of PPI inhibitors is still in demand or desirable. We report herein a simple method based on protein complementation for the in vitro study of PPIs, as well as screening of inhibitors against the PPI of interest. We have validated this system utilizing bacterial transcription factors NusB and NusE. Three derivatives of an inhibitor targeting the NusB-NusE interaction were synthesized and characterized with the system, which showed specific inhibition and antimicrobial activities. We have further confirmed the system with the RNA polymerase-σ interaction and an inhibitor. This system is expected to be suitable for more extensive high throughput screening of large chemical libraries. Additionally, our vector system can be easily adapted to study other PPI pairs, followed by inhibitor screening for hit identification in the application of early stage drug discovery.

Discovery of a potent tyrosine kinase AXL inhibitor bearing the 3-((2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yl)amino)pyrazine core.

Starting from the recently launched FLT3/AXL multi-targeted inhibitor Gilteritinib (5), we conducted a side-chain ring closure medicinal chemistry approach leading to the identification of compound 15c as a highly potent AXL inhibitor in the biochemical and cellular anti-proliferative assays, with IC50 values of 1.2 and 0.3 nM, respectively. Compared with the reference compound 5, our new discovered AXL inhibitor 15c is more potent in both assays.

Conjugation of Phthalocyanine Photosensitizer with Poly(amidoamine) Dendrimer: Improved Solubility, Disaggregation and Photoactivity Against HepG2 Cells.

OBJECTIVE: To improve solubility and to reduce aggregation, ZnPcC4 was conjugated to a third-generation poly-amidoamine dendrimer with amino end group (G3-PAMAM-NH2), which acts as a novel photodynamic therapy (PDT) drug carrier system. METHODS: The phthalocyanines were synthesized by construction reaction. The nano drug was obtained from the conjugation of ZnPcC4 to G3-PAMAM-NH2, using EDC and NHS as coupling agents. The ZnPcC4@G3-PAMAM-NH2 conjugation was characterized by UV-Vis and MS. The 1O2 quantum yield of ZnPcC4@G3-PAMAM-NH2 in water was measured by the chemiluminescence method. The in vitro PDT responses of the studied photosensitizers were studied in hepatocellular carcinoma cell line HepG2 by MTT assay. RESULTS: At ZnPcC4/G3-PAMAM-NH2 raw ratio of 100/1, the ZnPcC4 conjugate had improved solubility and reduced aggregation tendency in aqueous solution. At this optimum molar ratio, ZnPcC4- G3-PAMAM-NH2 inhibited HepG2 cells, with a half-maximal inhibitory concentration of 1.67 µg/mL upon infrared light exposure. The controls, including dark conditions, or media as well as G3-PAMAM-NH2 exposure, exhibited no inhibitory response. CONCLUSION: The conjugation of phthalocyanine photosensitizer ZnPcC4 to poly-amidoamine dendrimer G3-PAMAM-NH2 improved the PDT outcomes, in which the optimized binding ratio of ZnPcC4 to G3-PAMAM-NH2 was 6:1.

Genetic analysis of a morphologically heterogeneous ovarian endometrioid carcinoma.

AIMS: Low-grade ovarian endometrioid carcinomas may be associated with high-grade components. Whether the latter are clonally related to and originate from the low-grade endometrioid carcinoma remains unclear. The aim of this study was to use massively parallel sequencing to characterize the genomic landscape and clonal relatedness of an ovarian endometrioid carcinoma containing low-grade and high-grade components. METHODS AND RESULTS: DNA samples extracted from each tumour component (low-grade endometrioid, high-grade anaplastic and high-grade squamous) and matched normal tissue were subjected to targeted massively parallel sequencing with the 410-gene Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) sequencing assay. Somatic single nucleotide variants, small insertions and deletions, and copy number alterations were detected with state-of-the-art bioinformatics algorithms, and validated with orthogonal methods. The endometrioid carcinoma and the associated high-grade components shared copy number alterations and four clonal mutations, including SMARCA4 mutations, which resulted in loss of BRG1 protein expression. Subclonal mutations and mutations restricted to single components were also identified, such as distinct TP53 mutations restricted to each histological component. CONCLUSIONS: Histologically distinct components of ovarian endometrioid carcinomas may show intratumour genetic heterogeneity but be clonally related, harbouring a complex clonal composition. In the present case, SMARCA4 mutations were probably early events, whereas TP53 somatic mutations were acquired later in evolution.

Anti-inflammatory effect of the six compounds isolated from Nauclea officinalis Pierrc ex Pitard, and molecular mechanism of strictosamide via suppressing the NF-κB and MAPK signaling pathway in LPS-induced RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Nauclea officinalis Pierrc ex Pitard. is a Chinese medicinal herb that contains high level of alkaloids which is the most abundant and active constituent. Strictosamide isolated from Nauclea officinalis Pierrc ex Pitard. showed significant effects on inflammatory response, compared with pumiloside, 3-epi-pumiloside, vincosamide, 3α,5α-tetrahydrodeoxycordifoline lactam and naucleamide A-10-O-ß-D-glucopyranoside of this plant. AIM OF STUDY: we investigated the biological activities of the six compounds mentioned-above, and the underlying molecular mechanism exerted by the most potent one, strictosamide. MATERIALS AND METHODS: The effects of strictosamide and other five compounds on the inhibitory activity of nitric oxide (NO) were screened by Griess test. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in media were detected by using Enzyme-linked immunosorbent (ELISA) kits. The effects on the mRNA expression of nitric oxide synthase (iNOS), TNF-α and IL-1ß of strictosamide were further investigated by RT-qPCR. Western blot assay was conducted to illustrate the effects of strictosamide on iNOS and phosphorylation of p65, inhibitor of NF-κB (IκB)-α, IκB-kinase (IKK)-α as well as p-extracellular signal-regulated kinase (ERK), p-c-jun N-terminal kinase (JNK) and p-p38 in the protein levels. RESULTS: Strictosamide potently suppressed the productions of NO, TNF-α and IL-1ß in LPS-induced RAW 264.7 macrophages, and it dose-dependently alleviated the LPS-simulated protein level of iNOS as well as the mRNA expressions of iNOS, TNF-α and IL-1ß. In addition, molecular data revealed that strictosamide markedly decreased the expressions of p-p65, p-IκBα and p-IKKα. Furthermore, strictosamide significantly attenuated LPS-induced the phosphorylation of p38, ERK and JNK. CONCLUSIONS: At present study, the results indicated that the anti-inflammatory activity of strictosamide was associated with the restraint of NO, TNF-α and IL-1ß via negative regulation of both NF-κB and mitogen-activated protein kinases (MAPKs) in LPS-induced RAW 264.7 cells.

Enhanced fluorescence of tetrasulfonated zinc phthalocyanine by graphene quantum dots and its application in molecular sensing/imaging.

When excited at 435 nm, tetra-sulfonate zinc phthalocyanine (ZnPcS4 ) emitted dual fluorescence at 495 and 702 nm. The abnormal fluorescence at 495 nm was experimentally studied and analyzed in detail for the first time. The abnormal fluorescence at 495 nm was deduced to originate from triplet-triplet (T-T) energy transfer of excited phthalocyanine (3 *ZnPcS4 ). Furthermore, graphene quantum dots (GQDs) enhanced the 495 nm fluorescence quantum yield (Q) of ZnPcS4 . The fluorescence properties of ZnPcS4 -GQDs conjugate were retained in a cellular environment. Based on the fluorescence of ZnPcS4 -GQDs conjugate, we designed and prepared an Apt29/thrombin/Apt15 sandwich thrombin sensor with high specificity and affinity. This cost-saving, simple operational sensing strategy can be extended to use in sensing/imaging of other biomolecules.

Design and discovery of 4-anilinoquinazoline-urea derivatives as dual TK inhibitors of EGFR and VEGFR-2.

EGFR and VEGFR-2 are involved in pathological disorders and the progression of different kinds of tumors, the combined blockade of EGFR and VEGFR signaling pathways appears to be an attractive approach to cancer therapy. In this work, a series of 4-anilinoquinazoline derivatives containing substituted diaryl urea or glycine methyl ester moiety were designed and identified as EGFR and VEGFR-2 dual inhibitors. Compounds 19i, 19j and 19l exhibited the most potent inhibitory activities against EGFR (IC50 = 1 nM, 78 nM and 51 nM, respectively) and VEGFR-2 (IC50 = 79 nM, 14 nM and 14 nM, respectively), they showed good antiproliferative activities as well. Molecular docking established the interaction of 19i with the DFG-out conformation of VEGFR-2, suggesting that they might be type II kinase inhibitors.

Nauclea officinalis inhibits inflammation in LPS-mediated RAW 264.7 macrophages by suppressing the NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Nauclea officinalis has been traditionally used in China for the treatment of fever, pneumonia and enteritidis etc. This study aims to investigate effects of N. officinalis on the inflammatory response as well as the possible molecular mechanism in LPS-stimulated RAW 264.7 murine macrophage cells. MATERIALS AND METHODS: Anti-inflammatory activity of N. officinalis (10, 20, 50, and 100µg/mL) was investigated by using LPS-induced RAW 264.7 macrophages. The NO production was determined by assaying nitrite in culture supernatants with the Griess reagent. The levels of TNF-α, IL-6 and IL-1ß in culture media were measured with ELISA kits. Real time fluorescence quantitative PCR was detected for mRNA expression of iNOS, TNF-α, IL-6 and IL-1ß. Western blot assay was performed to illustrate the inhibitory effects of N. officinalis on phosphorylation of IκB-α and NF-κB p65. RESULTS: Treatment with N. officinalis (10-100µg/mL) dose-dependently inhibited the production as well as mRNA expression of NO, TNF-α, IL-6 and IL-1ß in RAW 264.7 macrophages. Western blot assay suggested that the mechanism of the anti-inflammatory effect was associated with the inhibition of phosphorylation of IκB-α and NF-κB p65. CONCLUSIONS: The results indicated that N. officinalis potentially inhibited the activation of upstream mediator NF-κB signaling pathway via suppressing phosphorylation of IκB-α and NF-κB p65 to inhibit LPS-stimulated inflammation.