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.

Polyketides with potential bioactivities from the mangrove-derived fungus Talaromyces sp. WHUF0362.

Metabolites of microorganisms have long been considered as potential sources for drug discovery. In this study, five new depsidone derivatives, talaronins A-E (1-5) and three new xanthone derivatives, talaronins F-H (6-8), together with 16 known compounds (9-24), were isolated from the ethyl acetate extract of the mangrove-derived fungus Talaromyces species WHUF0362. The structures were elucidated by analysis of spectroscopic data and chemical methods including alkaline hydrolysis and Mosher's method. Compounds 1 and 2 each attached a dimethyl acetal group at the aromatic ring. A putative biogenetic relationship of the isolated metabolites was presented and suggested that the depsidones and the xanthones probably had the same biosynthetic precursors such as chrysophanol or rheochrysidin. The antimicrobial activity assay indicated that compounds 5, 9, 10, and 14 showed potent activity against Helicobacter pylori with minimum inhibitory concentration (MIC) values in the range of 2.42-36.04 µmol/L. While secalonic acid D (19) demonstrated significant antimicrobial activity against four strains of H. pylori with MIC values in the range of 0.20 to 1.57 µmol/L. Furthermore, secalonic acid D (19) exhibited cytotoxicity against cancer cell lines Bel-7402 and HCT-116 with IC50 values of 0.15 and 0.19 µmol/L, respectively. The structure-activity relationship of depsidone derivatives revealed that the presence of the lactone ring and the hydroxyl at C-10 was crucial to the antimicrobial activity against H. pylori. The depsidone derivatives are promising leads to inhibit H. pylori and provide an avenue for further development of novel antibiotics. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00170-5.

Aculeaxanthones A-E, new xanthones from the marine-derived fungus Aspergillus aculeatinus WHUF0198.

Introduction: Dimeric natural products are widespread in plants and microorganisms, which usually have complex structures and exhibit greater bioactivities than their corresponding monomers. In this study, we report five new dimeric tetrahydroxanthones, aculeaxanthones A-E (4-8), along with the homodimeric tetrahydroxanthone secalonic acid D (1), chrysoxanthones B and C (2 and 3), and 4-4'-secalonic acid D (9), from different fermentation batches of the title fungus. Methods: A part of the culture was added to a total of 60 flasks containing 300 ml each of number II fungus liquid medium and culture 4 weeks in a static state at 28˚C. The liquid phase (18 L) and mycelia was separated from the fungal culture by filtering. A crude extract was obtained from the mycelia by ultrasound using acetone. To obtain a dry extract (18 g), the liquid phase combined with the crude extract were further extracted by EtOAc and concentrated in vacuo. The MIC of anaerobic bacteria was examined by a broth microdilution assay. To obtain MICs for aerobic bacteria, the agar dilution streak method recommended in Clinical and Laboratory Standards Institute document (CLSI) M07-A10 was used. Compounds 1-9 was tested against the Bel-7402, A-549 and HCT-116 cell lines according to MTT assay. Results and Discussion: The structures of these compounds were elucidated on the base of 1D and 2D NMR and HR-ESIMS data, and the absolute configurations of the new xanthones 4-8 were determined by conformational analysis and time-dependent density functional theory-electronic circular dichroism (TDDFT-ECD) calculations. Compounds 1-9 were tested for cytotoxicity against the Bel-7402, A549, and HCT-116 cancer cell lines. Of the dimeric tetrahydroxanthone derivatives, only compound 6 provided cytotoxicity effect against Bel-7402 cell line (IC50, 1.96 µM). Additionally, antimicrobial activity was evaluated for all dimeric tetrahydroxanthones, including four Gram-positive bacteria including Enterococcus faecium ATCC 19434, Bacillus subtilis 168, Staphylococcus aureus ATCC 25923 and MRSA USA300; four Gram-negative bacteria, including Helicobacter pylori 129, G27, as well as 26,695, and multi drug-resistant strain H. pylori 159, and one Mycobacterium M. smegmatis ATCC 607. However, only compound 1 performed activities against H. pylori G27, H. pylori 26695, H. pylori 129, H. pylori 159, S. aureus USA300, and B. subtilis 168 with MIC values of 4.0, 4.0, 2.0, 2.0, 2.0 and 1.0 µg/mL, respectively.

Two New Austocystin Analogs from the Marine-Derived Fungus Aspergillus sp. WHUF05236.

Two new austocystin analogs, austocystin P (1) and austocystin Q (2), along with fourteen known compounds (3-16) were isolated from the fermentation extract of Aspergillus sp. WHUF05236. The planar structures of 1 and 2 were elucidated through 1D, 2D NMR and MS analyses. Their absolute configurations were determined by the time-dependent density functional (TDDFT)-ECD calculation. Compounds 3, 11, and 12 exhibited antimicrobial activities against Helicobacter pylori with MIC values ranging from 20.00 to 43.47 µM. Compounds 3, 6, and 7 showed cytotoxicities against the human colon cancer cell lines Hct-116 with IC50 values of 101.79, 65.46, and 36.72 µM, respectively.

A phosphopantetheinyl transferase gene restricted to Porphyromonas.

The phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of carrier proteins (CPs) from fatty acid synthases (FASs) in primary metabolism and from polyketide synthases (PKSs) and non-ribosomal polypeptide synthases (NRPSs) in secondary metabolism. Based on the conserved sequence motifs and substrate specificities, two types (AcpS-type and Sfp-type) of PPTases have been identified in prokaryotes. We present here that Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, harbors merely one PPTase, namely PptP. Complementation and gene deletion experiments clearly show that PptP can replace the function of Escherichia coli AcpS and is essential for the growth of P. gingivalis. Purified PptP transfers the 4-phosphopantetheine moiety of CoA to inactive apo-acyl carrier protein (ACP) to form holo-ACP, which functions as an active carrier of the acyl intermediates of fatty acid synthesis. Moreover, PptP exhibits broad substrate specificity, modifying all ACP substrates tested and catalyzing the transfer of coenzyme A (CoA) derivatives. The lack of sequence alignment with known PPTases together with phylogenetic analyses revealed PptP as a new class of PPTases. Identification of the new PPTase gene pptP exclusive in Porphyromonas species reveals a potential target for treating P. gingivalis infections.

KCNN4-mediated Ca2+/MET/AKT axis is promising for targeted therapy of pancreatic ductal adenocarcinoma.

As a member of the potassium calcium-activated channel subfamily, increasing evidence suggests that KCNN4 was associated with malignancies. However, the roles and regulatory mechanisms of KCNN4 in PDAC have been little explored. In this work, we demonstrated that the level of KCNN4 in PDAC was abnormally elevated, and the overexpression of KCNN4 was induced by transcription factor AP-1. KCNN4 was closely correlated with unfavorable clinicopathologic characteristics and poor survival. Functionally, we found that overexpression of KCNN4 promoted PDAC cell proliferation, migration and invasion. Conversely, the knockdown of KCNN4 attenuated the growth and motility of PDAC cells. In addition to these, knockdown of KCNN4 promoted PDAC cell apoptosis and led to cell cycle arrest in the S phase. In mechanistic investigations, RNA-sequence revealed that the MET-mediated AKT axis was essential for KCNN4, encouraging PDAC cell proliferation and migration. Collectively, these findings reveal a function of KCNN4 in PDAC and suggest it's an attractive therapeutic target and tumor marker. Our studies underscore a better understanding of the biological mechanism of KCNN4 in PDAC and suggest novel strategies for cancer therapy.

Helicobacter pylori FabX contains a [4Fe-4S] cluster essential for unsaturated fatty acid synthesis.

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa.

Dihydrotanshinone I Is Effective against Drug-Resistant Helicobacter pylori In Vitro and In Vivo.

Helicobacter pylori is a major global pathogen and has been implicated in gastritis, peptic ulcer, and gastric carcinoma. The efficacy of the extensive therapy of H. pylori infection with antibiotics is compromised by the development of drug resistance and toxicity toward human gut microbiota, which urgently demands novel and selective antibacterial strategies. The present study was mainly performed to assess the in vitro and in vivo effects of a natural herbal compound, dihydrotanshinone I (DHT), against standard and clinical H. pylori strains. DHT demonstrated effective antibacterial activity against H. pyloriin vitro (MIC50/90, 0.25/0.5 µg/ml), with no development of resistance during continuous serial passaging. Time-kill curves showed strong time-dependent bactericidal activity for DHT. Also, DHT eliminated preformed biofilms and killed biofilm-encased H. pylori cells more efficiently than the conventional antibiotic metronidazole. In mouse models of multidrug-resistant H. pylori infection, dual therapy with DHT and omeprazole showed in vivo killing efficacy superior to that of the standard triple-therapy approach. Moreover, DHT treatment induces negligible toxicity against normal tissues and exhibits a relatively good safety index. These results suggest that DHT could be suitable for use as an anti-H. pylori agent in combination with a proton pump inhibitor to eradicate multidrug-resistant H. pylori.

Broad-Spectrum Antiviral Natural Products from the Marine-Derived Penicillium sp. IMB17-046.

A new pyrazine derivative, trypilepyrazinol (1), a new α-pyrone polyketide, (+)-neocitreoviridin (2), and a new ergostane analogue, 3ß-hydroxyergosta-8,14,24(28)-trien-7-one (3), were isolated and characterized along with five known compounds from the marine-derived fungus Penicillium sp. IMB17-046. The structures of these new compounds were determined using spectroscopic data analyses (HRESIMS, 1D- and 2D-NMR), X-ray crystallography analysis, and TDDFT ECD calculation. Compounds 1 and 3 exhibited broad-spectrum antiviral activities against different types of viruses, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza A virus (IAV), with IC50 values ranging from 0.5 to 7.7 µM. Compounds 1 and 2 showed antibacterial activities against Helicobacter pylori, a causative pathogen of various gastric diseases, with minimum inhibitory concentration (MIC) values of 1-16 µg/mL.

The Cyclopropane Fatty Acid Synthase Mediates Antibiotic Resistance and Gastric Colonization of Helicobacter pylori.

Cyclopropane fatty acids (CFAs) are synthetized by the addition of a methylene group from S-adenosyl-l-methionine across the carbon-carbon double bonds of unsaturated fatty acid chains of membrane phospholipids. This fatty acid cyclopropanation, catalyzed by the CFA synthase (CfaS) enzyme, occurs in many bacteria, including the human pathogen Helicobacter pylori Although the cyclopropane modification was reported to play a key role in the adaptation in response to environmental stress, its role in H. pylori remains unknown. In this study, we showed that H. pylori HP0416 encodes a functional CfaS. The enzyme was demonstrated to be required for acid resistance, antibiotic resistance, intracellular survival and mouse gastric colonization, and cell membrane integrity. Moreover, the tool compound dioctylamine, which acts as a substrate mimic, directly inhibits the CfaS function of H. pylori, resulting into sensitivity to acid stress, increased antibiotic susceptibility, and attenuated abilities to avoid macrophage killing and to colonize mouse stomachs. These results validate CfaS as a promising antibiotic target and provide new potentials for this recognized target in future anti-H. pylori drug discovery efforts.IMPORTANCE The increasing prevalence of multidrug-resistant Helicobacter pylori strains has created an urgent need for alternative therapeutic regimens that complement the current antibiotic treatment strategies for H. pylori eradication; however, this is greatly hampered due to a lack of "druggable" targets. Although the CFAs are present in H. pylori cytoplasmic membranes at high levels, their physiological role has not been established. In this report, deletion of the CFA synthase CfaS was shown to attenuate acid and drug resistance, immune escape, and gastric colonization of H. pylori These findings were validated by inhibition of the CfaS activity with the tool compound dioctylamine. These studies identify this enzyme as an attractive target for further drug discovery efforts against H. pylori.

In Vitro and In Vivo Activities of Zinc Linolenate, a Selective Antibacterial Agent against Helicobacter pylori.

Helicobacter pylori is a major global pathogen, and its infection represents a key factor in the etiology of various gastric diseases, including gastritis, peptic ulcers, and gastric carcinoma. The efficacy of current standard treatment for H. pylori infection including two broad-spectrum antibiotics is compromised by toxicity toward the gut microbiota and the development of drug resistance, which will likely only be resolved through novel and selective antibacterial strategies. Here, we synthesized a small molecule, zinc linolenate (ZnLla), and investigated its therapeutic potential for the treatment of H. pylori infection. ZnLla showed effective antibacterial activity against standard strains and drug-resistant clinical isolates of H. pyloriin vitro with no development of resistance during continuous serial passaging. The mechanisms of ZnLla action against H. pylori involved the disruption of bacterial cell membranes and generation of reactive oxygen species. In mouse models of multidrug-resistant H. pylori infection, ZnLla showed in vivo killing efficacy comparable and superior to the triple therapy approach when use as a monotherapy and a combined therapy with omeprazole, respectively. Moreover, ZnLla treatment induces negligible toxicity against normal tissues and causes minimal effects on both the diversity and composition of the murine gut microbiota. Thus, the high degree of selectivity of ZnLla for H. pylori provides an attractive candidate for novel targeted anti-H. pylori treatment.

Unsaturated Fatty Acid Synthesis in the Gastric Pathogen Helicobacter pylori Proceeds via a Backtracking Mechanism.

Helicobacter pylori is a Gram-negative bacterium that inhabits the upper gastrointestinal tract in humans, and the presence of this pathogen in the gut microbiome increases the risk of peptic ulcers and stomach cancer. H. pylori depends on unsaturated fatty acid (UFA) biosynthesis for maintaining membrane structure and function. Although some of the H. pylori enzymes involved in UFA biosynthesis are functionally homologous with the enzymes found in Escherichia coli, we show here that an enzyme HP0773, now annotated as FabX, uses an unprecedented backtracking mechanism to not only dehydrogenate decanoyl-acyl carrier protein (ACP) in a reaction that parallels that of acyl-CoA dehydrogenase, the first enzyme of the fatty acid ß-oxidation cycle, but also isomerizes trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, the key UFA synthetic intermediate. Thus, FabX reverses the normal fatty acid synthesis cycle in H. pylori at the C10 stage. Overall, this unusual FabX activity may offer a broader explanation for how many bacteria that lack the canonical pathway enzymes produce UFA-containing phospholipids.

Comparative Genomics of H. pylori and Non-Pylori Helicobacter Species to Identify New Regions Associated with Its Pathogenicity and Adaptability.

The genus Helicobacter is a group of Gram-negative, helical-shaped pathogens consisting of at least 36 bacterial species. Helicobacter pylori (H. pylori), infecting more than 50% of the human population, is considered as the major cause of gastritis, peptic ulcer, and gastric cancer. However, the genetic underpinnings of H. pylori that are responsible for its large scale epidemic and gastrointestinal environment adaption within human beings remain unclear. Core-pan genome analysis was performed among 75 representative H. pylori and 24 non-pylori Helicobacter genomes. There were 1173 conserved protein families of H. pylori and 673 of all 99 Helicobacter genus strains. We found 79 genome unique regions, a total of 202,359bp, shared by at least 80% of the H. pylori but lacked in non-pylori Helicobacter species. The operons, genes, and sRNAs within the H. pylori unique regions were considered as potential ones associated with its pathogenicity and adaptability, and the relativity among them has been partially confirmed by functional annotation analysis. However, functions of at least 54 genes and 10 sRNAs were still unclear. Our analysis of protein-protein interaction showed that 30 genes within them may have the cooperation relationship.