Mechanism of action of bacterial toxins elevating the cAMP in host cells

Cyclic adenosine monophosphate (cAMP) is an universal second messenger that regulates a large number of molecular mechanisms inside the eukaryotic cell. The level of synthesized cAMP is tightly regulated by endogenous adenylatecyclase (AC), and therefore this enzyme is often a target for various bacterial toxins. To manipulate intracellular cAMP levels in a target cell, bacteria have developed two different strategies for their toxins. Bordetella pertussis adenylate cyclase toxin (CyaA), Bacillus anthracis edema factor (EF) and Pseudomonas aeruginosa exotoxinY have in their structure an enzymatic AC domain which is activated by an intracellular cofactor and has several times higher activity than the eukaryotic AC enzyme itself. Other toxins, such as Bordetella pertussis pertussis toxin (PT), Vibrio cholerae cholera toxin (CT), and Escherichia coli heat labile toxin use ADP-ribosylation reaction of AC-coupled heterotrimeric G proteins to increase its activity and uncontrolled cAMP production. This work presents a literature search with accent on the molecular mechanism of interaction of these toxins with the target cell. Keywords: bacterial pathogens, virulence factors, intracellular cAMP elevation, bacterial toxins, adenylatecyclase (adenylylcyclase), Bordetella pertussis, Vibrio cholerae,...Cyklický adenosinmonofosfát (cAMP) je univerzálním druhým poslem, který reguluje velké množství molekulárních mechanismů uvnitř buňky. Hladina syntetizovaného cAMP je přísně regulována vnitrobuněčnou adenylátcyklázou (AC), a proto tento enzym často představuje cíl pro různé bakteriální toxiny. Pro manipulaci s hladinou vnitrobuněčného cAMP v cílové buňce vyvinuly bakterie pro své toxiny dvě odlišné strategie. Adenylátcyklázový toxin (CyaA) bakterie Bordetella pertussis, edemogenní faktor (EF) bakterie Bacillus anthracis a ExotoxinY bakterie Pseudomonas aeruginosa mají ve své struktuře enzymatickou AC doménu, která má po aktivaci intracelulárním kofaktorem několikrát vyšší aktivitu než vlastní eukaryotní adenylátcykláza, nacházející se uvnitř hostitelské buňky. Jiné toxiny, jako pertusový toxin (PT) bakterie Bordetella pertussis, cholerový toxin (CT) bakterie Vibrio cholerae a tepelně labilní toxin bakterie Escherichia coli využívají ADP-ribosylaci heterotrimerních G proteinů spřažených s vnitrobuněčnou AC pro zvýšení její aktivity a následnou nekontrolovanou produkci cAMP. Tato práce představuje literární rešerši uvedených toxinů s důrazem na jejich molekulární mechanizmy interakce s hostitelskou buňkou. Klíčová slova: pathogenní bakterie, faktory virulence, vnitrobuněčná hladina cAMP, bakteriální...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc

Sequence variation in CYP51A from the Y strain of Trypanosoma cruzi alters its sensitivity to inhibition

CYP51 (sterol 14α-demethylase) is an efficient target for clinical and agricultural antifungals and an emerging target for treatment of Chagas disease, the infection that is caused by multiple strains of a protozoan pathogen Trypanosoma cruzi. Here, we analyze CYP51A from the Y strain T. cruzi. In this protein, proline 355, a residue highly conserved across the CYP51 family, is replaced with serine. The purified enzyme retains its catalytic activity, yet has been found less susceptible to inhibition. These biochemical data are consistent with cellular experiments, both in insect and human stages of the pathogen. Comparative structural analysis of CYP51 complexes with VNI and two derivatives suggests that broad-spectrum CYP51 inhibitors are likely to be preferable as antichagasic drug candidates.Fil: Cherkesova, Tatiana S.. National Academy of Sciences of Belarus. Institute of Bioorganic Chemistry; BielorrusiaFil: Hargrove, Tatiana Y.. Vanderbilt University; Estados UnidosFil: Vanrell, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Ges, Igor. Vanderbilt University; Estados UnidosFil: Usanov, Sergey A.. National Academy of Sciences of Belarus. Institute of Bioorganic Chemistry; BielorrusiaFil: Romano, Patricia Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Cienicas Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lepesheva, Galina I.. Vanderbilt University; Estados Unido

The Antifungal Drug Isavuconazole Inhibits the Replication of Human Cytomegalovirus (HCMV) and Acts Synergistically with Anti-HCMV Drugs

We recently reported that some clinically approved antifungal drugs are potent inhibitors of human cytomegalovirus (HCMV). Here, we report the broad-spectrum activity against HCMV of isavuconazole (ICZ), a new extended-spectrum triazolic antifungal drug. ICZ inhibited the replication of clinical isolates of HCMV as well as strains resistant to the currently available DNA polymerase inhibitors. The antiviral activity of ICZ against HCMV could be linked to the inhibition of human cytochrome P450 51 (hCYP51), an enzyme whose activity we previously demonstrated to be required for productive HCMV infection. Moreover, time-of-addition studies indicated that ICZ might have additional inhibitory effects during the first phase of HCMV replication. Importantly, ICZ showed synergistic antiviral activity in vitro when administered in combination with different approved anti-HCMV drugs at clinically relevant doses. Together, these results pave the way to possible future clinical studies aimed at evaluating the repurposing potential of ICZ in the treatment of HCMV-associated diseases

Variability of fertility indicators in Ayrshire firstcalf heifers under the influence of early lactation

The basic value for optimizing the reproduction of the herd in dairy cattle breeding is the ability of cows to procreate offspring - fertility. It is a complex feature, characterized by many indicators and due to a complex of factors. The goal of our work was to reveal and to study these indicators and factors. The studies were carried out on Ayrshire cows of the Megrega breeding farm (the Republic of Karelia). The average milk yield per cow is over 9,000 kg of milk per year. Fertility indicators were taken into account for 5 years according to the data of 3866 lactations. The following fertility indicators were analysed: the indifference period; the period of insemination; the service period; fertilization from the first insemination; conception index (number of inseminations per conception); early embryonic death. We have analysed the relationship of these indicators with age, the amount of milk yield in general for lactation and during the early lactation period. An increase in the level of milk yield of cows during the current lactation up to 10,000 kg of milk or more was accompanied by a decrease in fertilization after the first insemination from 78.1 to 33.6%, as well as a significant deterioration in other main indicators of reproduction. The study established the relationship between the level of productivity of first-calf heifers during the early lactation period and fertility indicators. The greatest influence on fertility indicators during the early lactation period and insemination was exerted by the level of milk yield in the 1st month of the first lactation. The worst reproductive abilities were shown by first-calf heifers with an average daily milk yield of 34 kg or more. The results obtained can be used in further research on the development of selection indices, selection according to which can provide a high genetic trend in milk yield while maintaining and developing the progressive fertility of dairy cows

Nitrotriazole-based acetamides and propanamides with broad spectrum antitrypanosomal activity.

3-Nitro-1H-1,2,4-triazole-based acetamides bearing a biphenyl- or a phenoxyphenyl moiety have shown remarkable antichagasic activity both in vitro and in an acute murine model, as well as substantial in vitro antileishmanial activity but lacked activity against human African trypanosomiasis. We have shown now that by inserting a methylene group in the linkage to obtain the corresponding propanamides, both antichagasic and in particular anti-human African trypanosomiasis potency was increased. Therefore, IC50 values at low nM concentrations against both T. cruzi and T. b. rhodesiense, along with huge selectivity indices were obtained. Although several propanamides were active against Leishmania donovani, they were slightly less potent than their corresponding acetamides. There was a good correlation between lipophilicity (clogP value) and trypanocidal activity, for all new compounds. Type I nitroreductase, an enzyme absent from the human host, played a role in the activation of the new compounds, which may function as prodrugs. Antichagasic activity in vivo was also demonstrated with representative propanamides.This work was supported in part by internal funds of the Radiation Medicine Department at NorthShore University HealthSystem. In addition, the Drugs for Neglected Diseases initiative (DNDi) received financial support from the Bill & Melinda Gates Foundation (BMGF) to perform the in vitro screenings against parasites

Novel 3-nitrotriazole-based amides and carbinols as bifunctional anti-Chagasic agents.

3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based fluconazole analogs were synthesized as dual functioning antitrypanosomal agents. Such compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion of trypanosomatids and, at the same time, act as inhibitors of the sterol 14α-demethylase (T. cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to monotherapy, we believe that this emerging class of bifunctional compounds may introduce a new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in vivo evaluation of such compounds is discussed.This work was supported in part by internal funds of the Radiation Medicine Department at NorthShore University HealthSystem. Experiments on T. cruzi CYP51 were funded by NIH (GM067871, to G.I.L.). In vitro screenings against parasites were funded by DNDi. For that project, DNDi received funding from the following donors: Department for Internationl Development (DFID), U.K.; Bill & Melinda Gates Foundation (BMGF), USA; Reconstruction Credit Institution-Federal Ministry of Education and Research (KfW-BMBF), Germany; and Directorate-General for International Cooperation (DGIS), The Netherlands. B.A.-V. acknowledges financial support by FONDECYT Postdoctorado 3130364

Sterol 14α-Demethylase as a Potential Target for Antitrypanosomal Therapy: Enzyme Inhibition and Parasite Cell Growth

SummarySterol 14α-demethylases (CYP51) serve as primary targets for antifungal drugs, and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands that demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors

Concerning P450 Evolution: Structural Analyses Support Bacterial Origin of Sterol 14α-Demethylases

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Lamb, D. C., Hargrove, T. Y., Zhao, B., Wawrzak, Z., Goldstone, J. V., Nes, W. D., Kelly, S. L., Waterman, M. R., Stegeman, J. J., & Lepesheva, G. I. Concerning P450 evolution: structural analyses support bacterial origin of sterol 14α-demethylases. Molecular Biology and Evolution, (2020): msaa260, doi:10.1093/molbev/msaa260.Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an “orphan” P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.The study was supported by National Institutes of Health (Grant No. R01 GM067871 to G.I.L.) and by a UK-USA Fulbright Scholarship and the Royal Society (to D.C.L.)

Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit

Enzyme sterol 14α-demethylase (CYP51) is a well-established target for anti-fungal therapy and is a prospective target for Chagas' disease therapy. We previously identified a chemical scaffold capable of delivering a variety of chemical structures into the CYP51 active site. In this work the binding modes of several second generation compounds carrying this scaffold were determined in high-resolution co-crystal structures with CYP51 of Mycobacterium tuberculosis. Subsequent assays against CYP51 in Trypanosoma cruzi, the agent of Chagas' disease, demonstrated that two of the compounds bound tightly to the enzyme. Both were tested for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. This compound is currently being evaluated in animal models of Chagas' disease. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability of a single amino acid residue at a critical position in the active site. Our work is aimed at rational design of potent and highly selective CYP51 inhibitors with potential to become therapeutic drugs. Drug selectivity to prevent host–pathogen cross-reactivity is pharmacologically important, because CYP51 is present in human host