Discovery of Novel Cytochrome bc1 Complex Inhibitor Based on Natural Product Neopeltolide

Background: Natural products (NPs) are important sources for the design new drugs and agrochemicals. Neopeltolide, a marine NP, has been identified as a potent Qo-site inhibitor of cytochrome bc1 complex. Methods: In this study, a series of neopeltolide derivatives were designed and synthesized by the simplification of its 14-membered macrolactone ring with a diphenyl ether fragment. The enzymatic inhibition bioassays and mycelium growth inhibition experiments against a range of fungi were performed to determine their fungicidal activities. Results: The derivatives have potent activity against porcine bc1 complex. Compound 8q showed the best activity with an IC50 value of 24.41 nM, which was 8-fold more effective than that of positive control azoxystrobin. Compound 8a exhibited 100% inhibitory rate against Zymoseptoria tritici and Alternaria solani at a 20 mg/L dose. Conclusion: Computational results indicated that compounds with suitable physicochemical properties, as well as those forming a hydrogen bond with His161, would have good fungicidal activity. These data could be useful for the design of bc1 complex inhibitors in the future.

Study of Metyltetraprole, an unusual agrofungicide targeting the Qo-site of cytochrome bc1 complex

The mitochondrial respiratory chain bc1 complex is a proven target of agrofungicides. Most of them are Qo-site antagonists (i.e QoIs), competing with the substrate ubiquinol, and likely share the same binding mode as the widespread Qo-site resistance mutation G143A confers cross-resistance. Metyltetraprole (MTP) presents an exception as studies with phytopathogenic fungi showed that the inhibitor was unaffected by G143A. Here, we used the yeast model to investigate its mode of action. Analysis of bc1 complex mutants supports a Qo-site binding for MTP. However the compound seems distinct to other QoIs, such as azoxystrobin, in various ways, namely; 1) G143A was without effect on MTP, as previously reported. 2) The level of MTP resistance of mutants was higher in bc1 complex activity assays than in growth assays while the opposite was observed with azoxystrobin. 3) Steady-state kinetics used to characterise the mode of action of MTP also revealed differences compared to other QoIs.

Cryo-EM structure of the Rhodobacter sphaeroides RC-LH1 core monomer complex at 2.5 Å

Reaction centre light-harvesting 1 (RC-LH1) complexes are the essential components of bacterial photosynthesis. The membrane-intrinsic LH1 complex absorbs light and the energy migrates to an enclosed RC where a succession of electron and proton transfers conserves the energy as a quinol, which is exported to the cytochrome bc1 complex. In some RC-LH1 variants quinols can diffuse through small pores in a fully circular, 16-subunit LH1 ring, while in others missing LH1 subunits create a gap for quinol export. We used cryogenic electron microscopy to obtain a 2.5 Å resolution structure of one such RC-LH1, a monomeric complex from Rhodobacter sphaeroides. The structure shows that the RC is partly enclosed by a 14-subunit LH1 ring in which each αβ heterodimer binds two bacteriochlorophylls and, unusually for currently reported complexes, two carotenoids rather than one. Although the extra carotenoids confer an advantage in terms of photoprotection and light harvesting, they could block small pores in the LH1 ring and impede passage of quinones, necessitating a mechanism to create a dedicated quinone channel. The structure shows that two transmembrane proteins play a part in stabilizing an open ring structure; one of these components, the PufX polypeptide, is augmented by a hitherto undescribed protein subunit we designate as protein-Y, which lies against the transmembrane regions of the thirteenth and fourteenth LH1α polypeptides. Protein-Y prevents LH1 subunits 11-14 adjacent to the RC QB site from bending inwards towards the RC and, with PufX preventing complete encirclement of the RC, this pair of polypeptides ensures unhindered

Cytochrome bc1-aa3 oxidase supercomplex as emerging and potential drug target against tuberculosis

: The cytochrome bc1-aa3 supercomplex plays an essential role in the cellular respiratory system of Mycobacterium Tuberculosis. It transfers electrons from menaquinol to cytochrome aa3 (Complex IV) via cytochrome bc1 (Complex III), which reduces the oxygen. The electron transfer from a variety of donors into oxygen through the respiratory electron transport chain is essential to pump protons across the membrane creating an electrochemical transmembrane gradient (proton motive force, PMF) that regulates the synthesis of ATP via the oxidative phosphorylation process. Cytochrome bc1-aa3 supercomplex in M. tuberculosis is, therefore, a major drug target for antibiotic action. In recent years, several respiratory chain components have been targeted for developing new candidate drugs, illustrating the therapeutic potential of obstructing energy conversion of M. tuberculosis. The recently available cryo-EM structure of mycobacterial cytochrome bc1-aa3 supercomplex with open and closed conformations has opened new avenues for understanding its structure and function for developing more effective, new therapeutics against pulmonary tuberculosis. In this review, we discuss the role and function of several components, subunits, and drug targeting elements of the supercomplex cytochrome bc1-aa3, and its potential inhibitors in detail.

Cryo-EM structure of the Rhodospirillum rubrum RC-LH1 complex at 2.5 Å

The reaction centre light-harvesting 1 (RC-LH1) complex is the core functional component of bacterial photosynthesis. We determined the cryo-electron microscopy (cryo-EM) structure of the RC-LH1 complex from Rhodospirillum rubrum at 2.5 Å resolution, which reveals a unique monomeric bacteriochlorophyll with a phospholipid ligand in the gap between RC and LH1 complexes. The LH1 complex comprises a circular array of 16 αβ-polypeptide subunits that completely surrounds the RC, with a preferential binding site for a quinone, designated QP, on the inner face of the encircling LH1 complex. Quinols, initially generated at the RC QB site, are proposed to transiently occupy the QP site prior to traversing the LH1 barrier and diffusing to the cytochrome bc1 complex. Thus, the QP site, which is analogous to other such sites in recent cryo-EM structures of RC-LH1 complexes, likely reflects a general mechanism for exporting quinols from the RC-LH1 complex.

Oral Microbiota Composition and Function Changes During Chronic Erythematous Candidiasis

Oral microbiota is constantly changing with the host state, whereas the oral microbiome of chronic erythematous candidiasis remains poorly understood. The aim of this study was to compare oral microbial signatures and functional profiling between chronic erythematous candidiasis and healthy subjects. Using shotgun metagenomic sequencing, we analyzed the microbiome in 12 chronic erythematous candidiasis, 12 healthy subjects, and 2 chronic erythematous candidiasis cured by antifungal therapy. We found that the salivary microbiota of chronic erythematous candidiasis was significantly different from that of healthy subjects. Among them, Rothia mucilaginosa and Streptococcus mitis were the most abundant disease-enriched species (Mann-Whitney U-test, P < 0.05). In addition, co-occurrence network analysis showed that C. albicans formed densely connected modules with oral bacterial species and was mainly positive connected to Streptococcus species. Furthermore, we investigated the functional potentials of the microbiome and identified a set of microbial marker genes associated with chronic erythematous candidiasis. Some of these genes enriching in chronic erythematous candidiasis are involved in eukaryotic ribosome, putative glutamine transport system, and cytochrome bc1 complex respiratory unit. Altogether, this study revealed the changes of oral microbial composition, the co-occurrence between C. albicans and oral bacteria, as well as the changes of microbial marker genes during chronic erythematous candidiasis, which provides evidence of oral microbiome as a target for the treatment and prevention of chronic erythematous candidiasis.

Search for Novel Lead Inhibitors of Yeast Cytochrome bc1, from Drugbank and COCONUT

In this work we introduce a novel filtering and molecular modeling pipeline based on a fingerprint and descriptor similarity procedure, coupled with molecular docking and molecular dynamics (MD), to select potential novel quoinone outside inhibitors (QoI) of cytochrome bc1 with the aim of determining the same or different chromophores to usual. The study was carried out using the yeast cytochrome bc1 complex with its docked ligand (stigmatellin), using all the fungicides from FRAC code C3 mode of action, 8617 Drugbank compounds and 401624 COCONUT compounds. The introduced drug repurposing pipeline consists of compound similarity with C3 fungicides and molecular docking (MD) simulations with final QM/MM binding energy determination, while aiming for potential novel chromophores and perserving at least an amide (R1HN(C=O)R2) or ester functional group of almost all up to date C3 fungicides. 3D descriptors used for a similarity test were based on the 280 most stable Padel descriptors. Hit compounds that passed fingerprint and 3D descriptor similarity condition and had either an amide or an ester group were submitted to docking where they further had to satisfy both Chemscore fitness and specific conformation constraints. This rigorous selection resulted in a very limited number of candidates that were forwarded to MD simulations and QM/MM binding affinity estimations by the ORCA DFT program. In this final step, stringent criteria based on (a) sufficiently high frequency of H-bonds; (b) high interaction energy between protein and ligand through the whole MD trajectory; and (c) high enough QM/MM binding energy scores were applied to further filter candidate inhibitors. This elaborate search pipeline led finaly to four Drugbank synthetic lead compounds (DrugBank) and seven natural (COCONUT database) lead compounds—tentative new inhibitors of cytochrome bc1. These eleven lead compounds were additionally validated through a comparison of MM/PBSA free binding energy for new leads against those obtatined for 19 QoIs.

Modeling the Energy Landscape of Side Reactions in the Cytochrome bc1 Complex

Much of the metabolic molecular machinery responsible for energy transduction processes in living organisms revolves around a series of electron and proton transfer processes. The highly redox active enzymes can, however, also pose a risk of unwanted side reactions leading to reactive oxygen species, which are harmful to cells and are a factor in aging and age-related diseases. Using extensive quantum and classical computational modeling, we here show evidence of a particular superoxide production mechanism through stray reactions between molecular oxygen and a semiquinone reaction intermediate bound in the mitochondrial complex III of the electron transport chain, also known as the cytochrome bc1 complex. Free energy calculations indicate a favorable electron transfer from semiquinone occurring at low rates under normal circumstances. Furthermore, simulations of the product state reveal that superoxide formed at the Qo-site exclusively leaves the bc1 complex at the positive side of the membrane and escapes into the intermembrane space of mitochondria, providing a critical clue in further studies of the harmful effects of mitochondrial superoxide production.

Structural Investigation and Molecular Modeling Studies of Strobilurin-Based Fungicides Active against the Rice Blast Pathogen Pyricularia oryzae

The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type Pyricularia oryzae, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of P. oryzae cytochrome bc1 complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome bc1 active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.