Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

Functionalization of Chlorotonils: Dehalogenil as Promising Lead Compound for In Vivo Application.

The natural product chlorotonil displays high potency against multidrug-resistant Gram-positive bacteria and Plasmodium falciparum. Yet, its scaffold is characterized by low solubility and oral bioavailability, but progress was recently made to enhance these properties. Applying late-stage functionalization, we aimed to further optimize the molecule. Previously unknown reactions including a sulfur-mediated dehalogenation were revealed. Dehalogenil, the product of this reaction, was identified as the most promising compound so far, as this new derivative displayed improved solubility and in vivo efficacy while retaining excellent antimicrobial activity. We confirmed superb activity against multidrug-resistant clinical isolates of Staphylococcus aureus and Enterococcus spp. and mature transmission stages of Plasmodium falciparum. We also demonstrated favorable in vivo toxicity, pharmacokinetics and efficacy in infection models with S. aureus. Taken together, these results identify dehalogenil as an advanced lead molecule.

Novel 6-hydroxybenzothiazol-2-carboxamides as potent and selective monoamine oxidase B inhibitors endowed with neuroprotective activity.

In neurodegenerative diseases, using a single molecule that can exert multiple effects to modify the disease may have superior activity over the classical "one molecule-one target" approach. Herein, we describe the discovery of 6-hydroxybenzothiazol-2-carboxamides as highly potent and selective MAO-B inhibitors. Variation of the amide substituent led to several potent compounds having diverse side chains with cyclohexylamide 40 displaying the highest potency towards MAO-B (IC50 = 11 nM). To discover new compounds with extended efficacy against neurotoxic mechanisms in neurodegenerative diseases, MAO-B inhibitors were screened against PHF6, R3 tau, cellular tau and α-synuclein (α-syn) aggregation. We identified the phenethylamide 30 as a multipotent inhibitor of MAO-B (IC50 = 41 nM) and α-syn and tau aggregation. It showed no cytotoxic effects on SH-SY5Y neuroblastoma cells, while also providing neuroprotection against toxicities induced by α-syn and tau. The evaluation of key physicochemical and in vitro-ADME properties revealed a great potential as drug-like small molecules with multitarget neuroprotective activity.

High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

Development and evaluation of 2,4-disubstituted-5-aryl pyrimidine derivatives as antibacterial agents.

Designing novel candidates as potential antibacterial scaffolds has become crucial due to the lack of new antibiotics entering the market and the persistent rise in multidrug resistance. Here, we describe a new class of potent antibacterial agents based on a 5-aryl-N2,N4-dibutylpyrimidine-2,4-diamine scaffold. Structural optimization focused on the 5-aryl moiety and the bioisosteric replacement of the side chain linker atom. Screening of the synthesized compounds focused on a panel of bacterial strains, including gram-positive Staphylococcus aureus strains (Newman MSSA, methicillin- and vancomycin-resistant), and the gram-negative Escherichia coli (ΔAcrB strain). Several compounds showed broad-spectrum antibacterial activity with compound 12, bearing a 4-chlorophenyl substituent, being the most potent among this series of compounds. This frontrunner compound revealed a minimum inhibitory concentration (MIC) value of 1 µg/mL against the S. aureus strain (Mu50 methicillin-resistant S. aureus/vancomycin-intermediate S. aureus) and an MIC of 2 µg/mL against other tested strains. The most potent derivatives were further tested against a wider panel of bacteria and evaluated for their cytotoxicity, revealing further potent activities toward Streptococcus pneumoniae, Enterococcus faecium, and Enterococcus faecalis. To explore the mode of action, compound 12 was tested in a macromolecule inhibition assay. The obtained data were supported by the safety profile of compound 12, which possessed an IC50 of 12.3 µg/mL against HepG2 cells. The current results hold good potential for a new class of extended-spectrum antibacterial agents.

Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.

Correction: Fries et al. Impact of Drug Administration Routes on the In Vivo Efficacy of the Natural Product Sorangicin A Using a Staphylococcus aureus Infection Model in Zebrafish Embryos. Int. J. Mol. Sci. 2023, 24, 12791.

The authors would like to make the following corrections to the original publication [...].

Hit optimization by dynamic combinatorial chemistry on Streptococcus pneumoniae energy-coupling factor transporter ECF-PanT.

Herein, we present the first application of target-directed dynamic combinatorial chemistry (tdDCC) to the whole complex of the highly dynamic transmembrane, energy-coupling factor (ECF) transporter ECF-PanT in Streptococcus pneumoniae. In addition, we successfully employed the tdDCC technique as a hit-identification and -optimization strategy that led to the identification of optimized ECF inhibitors with improved activity. We characterized the best compounds regarding cytotoxicity and performed computational modeling studies on the crystal structure of ECF-PanT to rationalize their binding mode. Notably, docking studies showed that the acylhydrazone linker is able to maintain the crucial interactions.

Clean Synthetic Strategies to Biologically Active Molecules from Lignin: A Green Path to Drug Discovery.

Deriving active pharmaceutical agents from renewable resources is crucial to increasing the economic feasibility of modern biorefineries and promises to alleviate critical supply-chain dependencies in pharma manufacturing. Our multidisciplinary approach combines research in lignin-first biorefining, sustainable catalysis, and alternative solvents with bioactivity screening, an in vivo efficacy study, and a structural-similarity search. The resulting sustainable path to novel anti-infective, anti-inflammatory, and anticancer molecules enabled the rapid identification of frontrunners for key therapeutic indications, including an anti-infective against the priority pathogen Streptococcus pneumoniae with efficacy in vivo and promising plasma and metabolic stability. Our catalytic methods provided straightforward access, inspired by the innate structural features of lignin, to synthetically challenging biologically active molecules with the core structure of dopamine, namely, tetrahydroisoquinolines, quinazolinones, 3-arylindoles and the natural product tetrahydropapaveroline. Our diverse array of atom-economic transformations produces only harmless side products and uses benign reaction media, such as tunable deep eutectic solvents for modulating reactivity in challenging cyclization steps.

New Genetically Engineered Derivatives of Antibacterial Darobactins Underpin Their Potential for Antibiotic Development.

Biosynthetic engineering of bicyclic darobactins, selectively sealing the lateral gate of the outer membrane protein BamA, leads to active analogues, which are up to 128-fold more potent against Gram-negative pathogens compared to native counterparts. Because of their excellent antibacterial activity, darobactins represent one of the most promising new antibiotic classes of the past decades. Here, we present a series of structure-driven biosynthetic modifications of our current frontrunner, darobactin 22 (D22), to investigate modifications at the understudied positions 2, 4, and 5 for their impact on bioactivity. Novel darobactins were found to be highly active against critical pathogens from the WHO priority list. Antibacterial activity data were corroborated by dissociation constants with BamA. The most active derivatives D22 and D69 were subjected to ADMET profiling, showing promising features. We further evaluated D22 and D69 for bioactivity against multidrug-resistant clinical isolates and found them to have strong activity.

Selective Activation of a TRPC6 Ion Channel Over TRPC3 by Metalated Type-B Polycyclic Polyprenylated Acylphloroglucinols.

Selective modulation of TRPC6 ion channels is a promising therapeutic approach for neurodegenerative diseases and depression. A significant advancement showcases the selective activation of TRPC6 through metalated type-B PPAP, termed PPAP53. This success stems from PPAP53's 1,3-diketone motif facilitating metal coordination. PPAP53 is water-soluble and as potent as hyperforin, the gold standard in this field. In contrast to type-A, type-B PPAPs offer advantages such as gram-scale synthesis, easy derivatization, and long-term stability. Our investigations reveal PPAP53 selectively binding to the C-terminus of TRPC6. Although cryoelectron microscopy has resolved the majority of the TRPC6 structure, the binding site in the C-terminus remained unresolved. To address this issue, we employed state-of-the-art artificial-intelligence-based protein structure prediction algorithms to predict the missing region. Our computational results, validated against experimental data, indicate that PPAP53 binds to the 777LLKL780-region of the C-terminus, thus providing critical insights into the binding mechanism of PPAP53.

Identification of HuR-RNA Interfering Compounds by Dynamic Combinatorial Chemistry and Fluorescence Polarization.

The RNA binding protein HuR regulates the post-transcriptional process of different oncogenes and tumor suppressor genes, and its dysregulation is linked with cancer. Thus, modulating the complex HuR-RNA represents a promising anticancer strategy. To search for novel HuR ligands able to interfere with the HuR-RNA complex, the protein-templated dynamic combinatorial chemistry (pt-DCC) method was utilized. The recombinant RRM1+2 protein construct, which contains essential domains for ligand-HuR binding and exhibits enhanced solubility and stability compared to the native protein, was used for pt-DCC. Seven acylhydrazones with over 80% amplification were identified. The binding of the fragments to HuR extracted from DCC was validated using STD-NMR, and molecular modeling studies revealed the ability of the compounds to bind HuR at the mRNA binding pocket. Notably, three compounds effectively interfered with HuR-RNA binding in fluorescence polarization studies, suggesting their potential as foundational compounds for developing anticancer HuR-RNA interfering agents.

Identification of inhibitors targeting the energy-coupling factor (ECF) transporters.

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. The central role of vitamin transport in the metabolism of bacteria and absence from humans make the ECF transporters an attractive target for inhibition with selective chemical probes. Here, we report on the identification of a promising class of inhibitors of the ECF transporters. We used coarse-grained molecular dynamics simulations on Lactobacillus delbrueckii ECF-FolT2 and ECF-PanT to profile the binding mode and mechanism of inhibition of this novel chemotype. The results corroborate the postulated mechanism of transport and pave the way for further drug-discovery efforts.

Association between acceptance of routine pregnancy vaccinations and COVID-19 vaccine uptake in pregnant patients.

OBJECTIVE: COVID-19 vaccination is a key approach to reduce morbidity and mortality in pregnant patients and their newborns. Anti-vaccine sentiment has recently increased with unclear impact on pregnant patients. We examined the association between acceptance of tetanus-diphtheria-acellular pertussis (Tdap) and influenza vaccines, considered to be routine pregnancy vaccines, and COVID-19 vaccine acceptance. Secondarily, we identified other predictors of COVID-19 vaccine uptake and described pregnancy outcomes in patients who were and were not vaccinated during pregnancy. METHODS: A retrospective cohort study of all patients who delivered at a single site from December 2020 - March 2022. Demographic, pregnancy, neonatal, and vaccination data were abstracted from the electronic medical record, which imports vaccine history from the California Immunization Registry. The relationship between influenza and Tdap vaccine acceptance, other baseline characteristics, and COVID-19 vaccine uptake was assessed using univariable and multivariable regression analysis. RESULTS: Of the 7857 patients who delivered during the study period, 4410 (56.1%) accepted the COVID-19 vaccine. Of those who received the COVID-19 vaccine, 3363 (97.6%) and 3049 (88.5%) received influenza and Tdap vaccines, respectively. Patients were more likely to receive the COVID-19 vaccine if they had advanced maternal age, obesity, Asian race, and private insurance. After adjustment for baseline differences, COVID vaccine acceptance was associated with receipt of Tdap (aOR 2.10, 95% CI 1.90-2.33) and influenza vaccines (aOR 2.83, 95% CI 2.55-3.14). There were no differences in preterm birth, low birthweight, and NICU admission between patients who received and did not receive the COVID-19 vaccine. CONCLUSION: Patients were more likely to accept COVID-19 vaccination if they received Tdap or influenza vaccinations. Older age, obesity, Asian race, and private insurance were independent predictors of vaccine uptake. Disparities in COVID-19 vaccination uptake bear further exploration to guide efforts in equitable and widespread vaccine distribution.

Development of low-cost and personalized external silicone breast prosthesis produced by additive manufacturing for women who have undergone mastectomy: A pilot study.

BACKGROUND: The aim of this study was to develop a low-cost and personalized method for external breast prosthesis production. METHODS: The projected light method was used for the acquisition of the 3D geometry of the left breast of a healthy 29-year-old woman, 69 kg and 1.69 m. The 3D modeling software Blender was used to make the prosthesis model and mold with adjustments to the model's mesh, such as smoothing, assigning thickness, and creating the walls of the prosthesis mold. Two counter-molds were created. The pieces were manufactured on the 3D printer Stella Lite 3 using polylactic acid filament. Finally, the silicone was pigmented, and the mold was filled. FINDINGS: Prototype 1 of the prosthesis was produced using a mold without a counter-mold, which resulted in a prosthesis of 495 g, considered heavy compared to traditional prostheses for the same breast size. To solve this issue, a counter-mold with pins was used to produce prototype 2 with a mass of 393 g, 20.6% lighter than prototype 1. Prototype 3 was made with a central-volume counter-mold and presented a mass of 355 g, a reduction of 28.3% compared to prototype 1. The definitive breast prosthesis was made with the pin counter-mold with a different silicone. It has nipple and areola pigmentation and a mass of 294 g, 25.2% lighter than prototype 2. INTERPRETATION: The results suggest that the projected light method and additive manufacturing are potential tools for developing external breast prostheses, which may improve the health conditions and quality of life of mastectomized women.

Thermoresponsive cholesteric liquid-crystal systems doped with terpenoids as drug delivery systems for skin applications.

Stimuli-responsive and tunable soft-matter systems are an advanced class of materials applicable for drug delivery. Liquid crystals (LCs) are promising candidates as multifunctional materials that can respond to temperature, light or magnetic field. Particularly, ordering and physical properties of thermoresponsive LCs depend predominantly on temperature as external trigger. The current work addresses an elegant strategy to implement the anisotropic properties of thermoresponsive LCs with a view to extending their application for drug delivery. We firstly fabricated novel compositions with a thermotropic core based on natural products - cholesteryl esters and mono-/bicyclic terpenoids. The distinctive feature of aforementioned systems is their temperature-induced switchability of drug release by transition to the LC state, depending on the skin temperature. Their mesomorphic and optical behavior was characterized via differential scanning calorimetry and polarizing optical microscopy. Furthermore, we describe the dependence of helical pitch on LC formulation for various ternary cholesteric systems doped with terpenoids, suggesting that these stimuli-responsive chiral dopants are nominally untwisting. Data from fluorescence probe technique indicate that cholesteryl esters and terpenoids as essential components of those LC systems jointly disrupt the tight structure of phospholipid bilayer packing enabling the facilitated penetration of drugs. The potential of LC formulations was explored for several model drugs with diverse physicochemical properties by in vitro and ex vivo penetration tests using artificial membranes and full human skin. Our findings confirm the potential of LC systems for various applications in skin drug delivery.

Exploring the Translational Gap of a Novel Class of Escherichia coli IspE Inhibitors.

Discovery of novel antibiotics needs multidisciplinary approaches to gain target enzyme and bacterial activities while aiming for selectivity over mammalian cells. Here, we report a multiparameter optimisation of a fragment-like hit that was identified through a structure-based virtual-screening campaign on Escherichia coli IspE crystal structure. Subsequent medicinal-chemistry design resulted in a novel class of E. coli IspE inhibitors, exhibiting activity also against the more pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. While cytotoxicity remains a challenge for the series, it provides new insights on the molecular properties for balancing enzymatic target and bacterial activities simultaneously as well as new starting points for the development of IspE inhibitors with a predicted new mode of action.

Impact of Drug Administration Routes on the In Vivo Efficacy of the Natural Product Sorangicin a Using a Staphylococcus aureus Infection Model in Zebrafish Embryos.

Staphylococcus aureus causes a wide range of infections, and it is one of the leading pathogens responsible for deaths associated with antimicrobial resistance, the rapid spread of which among S. aureus urges the discovery of new antibiotics. The evaluation of in vivo efficacy of novel drug candidates is usually performed using animal models. Recently, zebrafish (Danio rerio) embryos have become increasingly attractive in early drug discovery. Herein, we established a zebrafish embryo model of S. aureus infection for evaluation of in vivo efficacy of novel potential antimicrobials. A local infection was induced by microinjecting mCherry-expressing S. aureus Newman followed by treatment with reference antibiotics via microinjection into different injection sites as well as via waterborne exposure to study the impact of the administration route on efficacy. We successfully used the developed model to evaluate the in vivo activity of the natural product sorangicin A, for which common mouse models were not successful due to fast degradation in plasma. In conclusion, we present a novel screening platform for assessing in vivo activity at the antibiotic discovery stage. Furthermore, this work provides consideration for the choice of an appropriate administration route based on the physicochemical properties of tested drugs.

1-deoxy-D-xylulose-5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding.

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.