Selective killing of the human gastric pathogen Helicobacter pylori by mitochondrial respiratory complex I inhibitors.

Respiratory complex I is a multicomponent enzyme conserved between eukaryotic cells and many bacteria, which couples oxidation of electron donors and quinone reduction with proton pumping. Here, we report that protein transport via the Cag type IV secretion system, a major virulence factor of the Gram-negative bacterial pathogen Helicobacter pylori, is efficiently impeded by respiratory inhibition. Mitochondrial complex I inhibitors, including well-established insecticidal compounds, selectively kill H. pylori, while other Gram-negative or Gram-positive bacteria, such as the close relative Campylobacter jejuni or representative gut microbiota species, are not affected. Using a combination of different phenotypic assays, selection of resistance-inducing mutations, and molecular modeling approaches, we demonstrate that the unique composition of the H. pylori complex I quinone-binding pocket is the basis for this hypersensitivity. Comprehensive targeted mutagenesis and compound optimization studies highlight the potential to develop complex I inhibitors as narrow-spectrum antimicrobial agents against this pathogen.

Agrochemical Lessons for Infectious Disease Research: New Resistance Breaking Antifungal Hits against Candida auris.

Analysis of the history of the invention of the block-buster antifungal drug Fluconazole underscores the importance of agrochemical research on drug discovery and development. The multidrug resistant fungal pathogen Candida auris is now responsible for serious morbidity and mortality among immuno-compromised and long-term resident hospital patients globally. New drugs against C. auris are urgently needed. A focused screening of 1487 fungicides from the BASF agrochemical collection gave several potent inhibitors of C. auris with yet noncommercialized modes of action. The hits showed only minor activity loss against the azole-resistant C. auris strain CDC 0385 and low to moderate cytotoxicity to human HepG2 cells. Aminopyrimidine 4 showed high activity against resistant strains and selectivity in a HepG2 cells assay and is a potential hit candidate for further optimization.

Inhibition profile of trifludimoxazin towards PPO2 target site mutations.

BACKGROUND: Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO-inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone-type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO-inhibiting herbicides. RESULTS: Mutated Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations. CONCLUSION: Trifludimoxazin is a potent PPO-inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical-based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Inhibition of acyl-ACP thioesterase as site of action of the commercial herbicides cumyluron, oxaziclomefone, bromobutide, methyldymron and tebutam.

BACKGROUND: Understanding the mode and site of action of a herbicide is key for its efficient development, the evaluation of its toxicological risk, efficient weed control and resistance management. Recently, the mode of action (MoA) of the herbicide cinmethylin was identified in lipid biosynthesis with acyl-ACP thioesterase (FAT) as the site of action (SoA). Cinmethylin was registered for selective use in cereal crops for the control of grass weeds in 2020. RESULTS: Here, we present a high-resolution co-crystal structure of FAT in complex with cumyluron identified by a high throughput crystallization screen. We show binding to and inhibition of FAT by cumyluron. Furthermore, in an array of experiments consisting of FAT binding assays, FAT inhibition assays, physiological and metabolic profiling, we tested compounds that are structurally related to cumyluron and identified the commercial herbicides oxaziclomefone, methyldymron, tebutam and bromobutide, with so far unknown sites of action, as FAT inhibitors. Additionally, we show that the previously described FAT inhibitors cinmethylin and methiozolin bind to FAT in a nanomolar range, inhibit FAT enzymatic activity and lead to similar metabolic changes. CONCLUSION: Based on presented data, we corroborate cinmethylin and methiozolin as potent FAT inhibitors and identify FAT as the SoA of the herbicides cumyluron, oxaziclomefone, bromobutide, methyldymron and tebutam. © 2022 Society of Chemical Industry.

Targeting the IspD Enzyme in the MEP Pathway: Identification of a Novel Fragment Class.

The enzymes of the 2-C-methylerythritol-d-erythritol 4-phosphate (MEP) pathway (MEP pathway or non-mevalonate pathway) are responsible for the synthesis of universal precursors of the large and structurally diverse family of isoprenoids. This pathway is absent in humans, but present in many pathogenic organisms and plants, making it an attractive source of drug targets. Here, we present a high-throughput screening approach that led to the discovery of a novel fragment hit active against the third enzyme of the MEP pathway, PfIspD. A systematic SAR investigation afforded a novel chemical structure with a balanced activity-stability profile (16). Using a homology model of PfIspD, we proposed a putative binding mode for our newly identified inhibitors that sets the stage for structure-guided optimization.

Assessment of the rules related to gaining activity against Gram-negative bacteria.

In the search for new antibacterial compounds, we repositioned an antimalarial compound class by derivatising it based on the so-called "eNTRy" rules for enhanced accumulation into Gram-negative bacteria. We designed, synthesised and evaluated a small library of amino acid modified compounds together with the respective Boc-protected analogues, leading to no substantial improvement in antibacterial activity against Escherichia coli wild-type K12, whereas more distinct activity differences were observed in E. coli mutant strains ΔtolC, D22, ΔacrB and BL21(DE3)omp8. A comparison of the activity results of the E. coli mutants with respect to the known rules related to enhanced activity against Gram-negative bacteria revealed that applicability of the rules is not always ensured. Out of the four amino acids used in this study, glycine derivatives showed highest antibacterial activity, although still suffering from efflux issues.

Search for the Active Ingredients from a 2-Aminothiazole DMSO Stock Solution with Antimalarial Activity.

Chemical decomposition of DMSO stock solutions is a common incident that can mislead biological screening campaigns. Here, we share our case study of 2-aminothiazole 1, originating from an antimalarial class that undergoes chemical decomposition in DMSO at room temperature. As previously measured biological activities observed against Plasmodium falciparum NF54 and for the target enzyme PfIspE were not reproducible for a fresh batch, we tackled the challenge to understand where the activity originated from. Solvent- and temperature-dependent studies using HRMS and NMR spectroscopy to monitor the decomposition led to the isolation and in vitro evaluation of several fractions against PfIspE. After four days of decomposition, we successfully isolated the oxygenated and dimerised compounds using SFC purification and correlated the observed activities to them. Due to the unstable nature of the two isolates, it is likely that they undergo further decomposition contributing to the overall instability of the compound.

Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc1:aa3. While the cytochrome bc1:aa3 is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc1:aa3 to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.

A new herbicidal site of action: Cinmethylin binds to acyl-ACP thioesterase and inhibits plant fatty acid biosynthesis.

The prevalent occurrence of herbicide resistant weeds increases the necessity for new site of action herbicides for effective control as well as to relax selection pressure on the known sites of action. As a consequence, interest increased in the unexploited molecule cinmethylin as a new solution for the control of weedy grasses in cereals. Therefore, the mechanism of action of cinmethylin was reevaluated. We applied the chemoproteomic approach cellular Target Profiling™ from Evotec to identify the cinmethylin target in Lemna paucicostata protein extracts. We found three potential targets belonging to the same protein family of fatty acid thioesterases (FAT) to bind to cinmethylin with high affinity. Binding of cinmethylin to FAT proteins from Lemna and Arabidopsis was confirmed by fluorescence-based thermal shift assay. The plastid localized enzyme FAT plays a crucial role in plant lipid biosynthesis, by mediating the release of fatty acids (FA) from its acyl carrier protein (ACP) which is necessary for FA export to the endoplasmic reticulum. GC-MS analysis of free FA composition in Lemna extracts revealed strong reduction of unsaturated C18 as well as saturated C14, and C16 FAs upon treatment with cinmethylin, indicating that FA release for subsequent lipid biosynthesis is the primary target of cinmethylin. Lipid biosynthesis is a prominent target of different herbicide classes. To assess whether FAT inhibition constitutes a new mechanism of action within this complex pathway, we compared physiological effects of cinmethylin to different ACCase and VLCFA synthesis inhibitors and identified characteristic differences in plant symptomology and free FA composition upon treatment with the three herbicide classes. Also, principal component analysis of total metabolic profiling of treated Lemna plants showed strong differences in overall metabolic changes after cinmethylin, ACCase or VLCFA inhibitor treatments. Our results identified and confirmed FAT as the cinmethylin target and validate FAT inhibition as a new site of action different from other lipid biosynthesis inhibitor classes.

Potent Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT) Featuring a Spirocyclic Scaffold.

With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2-indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14-76 nm) and cellular potencies in the low nanomolar range (165-334 nm) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co-crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2-2.4 Šresolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non-spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor-bound ligands is discussed.

Conformational Aspects in the Design of Inhibitors for Serine Hydroxymethyltransferase (SHMT): Biphenyl, Aryl Sulfonamide, and Aryl Sulfone Motifs.

Malaria remains a major threat to mankind due to the perpetual emergence of resistance against marketed drugs. Twenty-one pyrazolopyran-based inhibitors bearing terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs were synthesized and tested towards serine hydroxymethyltransferase (SHMT), a key enzyme of the folate cycle. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target, as well as PfNF54 strains in cell-based assays in the low nanomolar range (18-56 nm). Seven co-crystal structures with P. vivax (Pv) SHMT were solved at 2.2-2.6 Šresolution. We observed an unprecedented influence of the torsion angle of ortho-substituted biphenyl moieties on cell-based efficacy. The peculiar lipophilic character of the sulfonyl moiety was highlighted in the complexes with aryl sulfonamide analogues, which bind in their preferred staggered orientation. The results are discussed within the context of conformational preferences in the ligands.

Mechanism of Allosteric Inhibition of the Enzyme IspD by Three Different Classes of Ligands.

Enzymes of the nonmevalonate pathway of isoprenoid biosynthesis are attractive targets for the development of herbicides and drugs against infectious diseases. While this pathway is essential for many pathogens and plants, mammals do not depend on it for the synthesis of isoprenoids. IspD, the third enzyme of the nonmevalonate pathway, is unique in that it has an allosteric regulatory site. We elucidated the binding mode of phenylisoxazoles, a new class of allosteric inhibitors. Allosteric inhibition is effected by large conformational changes of a loop region proximal to the active site. We investigated the different roles of residues in this loop by mutation studies and identified repulsive interactions with Asp291 and Asp292 to be responsible for inhibition. Crystallographic data and the response of mutant enzymes to three different classes of allosteric inhibitors provide an in-depth understanding of the allosteric mechanism. The obtained mutant enzymes show selective resistance to allosteric inhibitors and provide conceptually valuable information for future engineering of herbicide-resistant crops. We found that the isoprenoid precursors IPP and DMAPP are natural inhibitors of Arabidopsis thaliana IspD; however, they do not seem to bind to the allosteric site.

Antimalarial Inhibitors Targeting Serine Hydroxymethyltransferase (SHMT) with in Vivo Efficacy and Analysis of their Binding Mode Based on X-ray Cocrystal Structures.

Target-based approaches toward new antimalarial treatments are highly valuable to prevent resistance development. We report several series of pyrazolopyran-based inhibitors targeting the enzyme serine hydroxymethyltransferase (SHMT), designed to improve microsomal metabolic stability and to identify suitable candidates for in vivo efficacy evaluation. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target assays and PfNF54 strains in cell-based assays with values in the low nanomolar range (3.2-55 nM). A set of carboxylate derivatives demonstrated markedly improved in vitro metabolic stability (t1/2 > 2 h). A selected ligand showed significant in vivo efficacy with 73% of parasitemia reduction in a mouse model. Five new cocrystal structures with PvSHMT were solved at 2.3-2.6 Å resolution, revealing a unique water-mediated interaction with Tyr63 at the end of the para-aminobenzoate channel. They also displayed the high degree of conformational flexibility of the Cys364-loop lining this channel.

An amphiphilic graft copolymer-based nanoparticle platform for reduction-responsive anticancer and antimalarial drug delivery.

Medical applications of anticancer and antimalarial drugs often suffer from low aqueous solubility, high systemic toxicity, and metabolic instability. Smart nanocarrier-based drug delivery systems provide means of solving these problems at once. Herein, we present such a smart nanoparticle platform based on self-assembled, reduction-responsive amphiphilic graft copolymers, which were successfully synthesized through thiol-disulfide exchange reaction between thiolated hydrophilic block and pyridyl disulfide functionalized hydrophobic block. These amphiphilic graft copolymers self-assembled into nanoparticles with mean diameters of about 30-50 nm and readily incorporated hydrophobic guest molecules. Fluorescence correlation spectroscopy (FCS) was used to study nanoparticle stability and triggered release of a model compound in detail. Long-term colloidal stability and model compound retention within the nanoparticles was found when analyzed in cell media at body temperature. In contrast, rapid, complete reduction-triggered disassembly and model compound release was achieved within a physiological reducing environment. The synthesized copolymers revealed no intrinsic cellular toxicity up to 1 mg mL(-1). Drug-loaded reduction-sensitive nanoparticles delivered a hydrophobic model anticancer drug (doxorubicin, DOX) to cancer cells (HeLa cells) and an experimental, metabolically unstable antimalarial drug (the serine hydroxymethyltransferase (SHMT) inhibitor (±)-1) to Plasmodium falciparum-infected red blood cells (iRBCs), with higher efficacy compared to similar, non-sensitive drug-loaded nanoparticles. These responsive copolymer-based nanoparticles represent a promising candidate as smart nanocarrier platform for various drugs to be applied to different diseases, due to the biocompatibility and biodegradability of the hydrophobic block, and the protein-repellent hydrophilic block.

Aryl Bis-Sulfonamide Inhibitors of IspF from Arabidopsis thaliana and Plasmodium falciparum.

2-Methylerythritol 2,4-cyclodiphosphate synthase (IspF) is an essential enzyme for the biosynthesis of isoprenoid precursors in plants and many human pathogens. The protein is an attractive target for the development of anti-infectives and herbicides. Using a photometric assay, a screen of 40 000 compounds on IspF from Arabidopsis thaliana afforded symmetrical aryl bis-sulfonamides that inhibit IspF from A. thaliana (AtIspF) and Plasmodium falciparum (PfIspF) with IC50 values in the micromolar range. The ortho-bis-sulfonamide structural motif is essential for inhibitory activity. The best derivatives obtained by parallel synthesis showed IC50 values of 1.4 µm against PfIspF and 240 nm against AtIspF. Substantial herbicidal activity was observed at a dose of 2 kg ha(-1) . Molecular modeling studies served as the basis for an in silico search targeted at the discovery of novel, non-symmetrical sulfonamide IspF inhibitors. The designed compounds were found to exhibit inhibitory activities in the double-digit micromolar IC50 range.

Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities.

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Novel bioassay for the discovery of inhibitors of the 2-C-methyl-D-erythritol 4-phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis.

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl diphosphate in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisic acid and gibberellins. Consequently, disruption of this pathway is harmful to plants. We developed an in vivo bioassay that can measure the carbon flow through the carotenoid pathway. Leaf cuttings are incubated in the presence of a phytoene desaturase inhibitor to induce phytoene accumulation. Any compound reducing the level of phytoene accumulation is likely to interfere with either one of the steps in the MEP pathway or the synthesis of geranylgeranyl diphosphate. This concept was tested with known inhibitors of steps of the MEP pathway. The specificity of this in vivo bioassay was also verified by testing representative herbicides known to target processes outside of the MEP and carotenoid pathways. This assay enables the rapid screen of new inhibitors of enzymes preceding the synthesis of phytoene, though there are some limitations related to the non-specific effect of some inhibitors on this assay.

Deorphaning pyrrolopyrazines as potent multi-target antimalarial agents.

The discovery of pyrrolopyrazines as potent antimalarial agents is presented, with the most effective compounds exhibiting EC50 values in the low nanomolar range against asexual blood stages of Plasmodium falciparum in human red blood cells, and Plasmodium berghei liver schizonts, with negligible HepG2 cytotoxicity. Their potential mode of action is uncovered by predicting macromolecular targets through avant-garde computer modeling. The consensus prediction method suggested a functional resemblance between ligand binding sites in non-homologous target proteins, linking the observed parasite elimination to IspD, an enzyme from the non-mevalonate pathway of isoprenoid biosynthesis, and multi-kinase inhibition. Further computational analysis suggested essential P. falciparum kinases as likely targets of our lead compound. The results obtained validate our methodology for ligand- and structure-based target prediction, expand the bioinformatics toolbox for proteome mining, and provide unique access to deciphering polypharmacological effects of bioactive chemical agents.

Pseudilins: halogenated, allosteric inhibitors of the non-mevalonate pathway enzyme IspD.

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis have been identified as attractive targets with novel modes of action for the development of herbicides for crop protection and agents against infectious diseases. This pathway is present in many pathogenic organisms and plants, but absent in mammals. By using high-throughput screening, we identified highly halogenated marine natural products, the pseudilins, to be inhibitors of the third enzyme, IspD, in the pathway. Their activity against the IspD enzymes from Arabidopsis thaliana and Plasmodium vivax was determined in photometric and NMR-based assays. Cocrystal structures revealed that pseudilins bind to an allosteric pocket by using both divalent metal ion coordination and halogen bonding. The allosteric mode of action for preventing cosubstrate (CTP) binding at the active site was elucidated. Pseudilins show herbicidal activity in plant assays and antiplasmodial activity in cell-based assays.

In search of new herbicidal inhibitors of the non-mevalonate pathway.

The first five steps of the non-mevalonate pathway have been tested in high-throughput screening (HTS) campaigns, using enzymes of plant origin. Hit rates were in general relatively low, which could be attributed to the high polarity and charged nature of substrates and active sites of these enzymes. Still, for all the enzymes, apart from IspF (2-methylerythritol 2,4-cyclodiphosphate synthase), inhibitors could be identified with activities below 100 µM, and these were followed up to identify structure-activity relationships (SARs). For the enzyme IspD (2C-methyl-D-erythritol 4-phosphate cytidyltransferase), inhibitors with IC50 down to 35 nM were identified that also showed herbicidal activity.