Structural investigation of vitamin K epoxide reductase domain-containing protein in Leptospira species: a potential target for the development of new leptospirosis treatments as an alternative to antibiotics.

Leptospirosis is a worldwide zoonosis caused by the motile bacterium Leptospira. This disease can cause hemorrhagic symptoms, multi-visceral and renal failures, resulting in one million cases and approximately 60,000 deaths each year. The motility of Leptospira is highly involved in its virulence and is ensured by the presence of two flagella in the periplasm. Several proteins that require the formation of disulfide bridges are essential for flagellar function. In Leptospira, these redox reactions are catalysed by the vitamin K epoxide reductase domain-containing protein (VKORdcp). The aim of the present work was to study the conservation of VKORdcp among Leptospira species and its interactions with putative substrates and inhibitor. Our results evidenced the presence of ten amino acids specific to either pathogenic or saprophytic species. Furthermore, structural studies revealed a higher affinity of the enzyme for vitamin K1 quinone, compared to ubiquinone. Finally, characterisation of the binding of a potential inhibitor revealed the involvement of some VKORdcp amino acids that have not been present in the human enzyme, in particular the polar residue D114. Our study thus paves the way for the future development of Leptospira VKORdcp inhibitors, capable of blocking bacterial motility. Such molecules could therefore offer a promising therapeutic alternative to antibiotics, especially in the event of the emergence of antibiotic-resistant strains.Communicated by Ramaswamy H. Sarma.

Exposure and resistance to anticoagulant rodenticides in invasive and endemic Chadian urban rodent species to develop a rational management strategy.

Rodent management involves the use of anticoagulant rodenticides (ARs). This use has resulted in the selection of numerous resistance alleles in the Vkorc1 gene, encoding the target enzyme of ARs. In Africa, although rodents are a major problem as a consequence of their transport and transmission of zoonotic pathogens, and damage to crops, the use of ARs and the spread of resistance alleles are poorly documented. We attempted to address both issues in Chad which is one of the largest countries in Africa. Owing to its location at the crossroads of central and northern Africa, Chad is representative of many African countries. METHODS: Using a sampling of nearly 300 rodents composed of invasive and endemic rodents collected in six of Chad's largest cities, exposure to ARs was analyzed by their quantification in the liver; the spread of AR resistance alleles was analyzed by Vkorc1 sequencing. RESULTS: We demonstrate the use of both ARs generations in Chadian cities and report the total sequencing of the Vkorc1 for 44 Mastomys natalensis with detection of two different haplotypes, the sequencing of the Vkorc1 for two other endemic rodent species, M. kollmannspergeri and Arvicanthis niloticus, and finally the detection of three new missense mutations - V29E, V69E and D127V - in R. rattus, potentially associated with resistance to ARs. DISCUSSION: These results should argue for the implementation of a reasoned management of rodent populations in Africa to avoid the spread of ARs resistance alleles. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Design of a structure-activity relationship model of vitamin K epoxide reductase (VKORC1) inhibitors combining chemical synthesis of new compounds, enzymatic assays and molecular modelling.

Vitamin K antagonists (VKAs) anticoagulants have been used since the 1950s as medicines and rodenticides. These molecules are mainly 4-hydroxycoumarin derivatives and act by inhibiting the vitamin K epoxide reductase (VKORC1), an endoplasmic reticulum membrane resident enzyme. However, many VKORC1 mutations have been reported over the last decade, inducing VKAs resistances and thus treatments failures. Although studies have reported experimental and computational investigations of VKAs based on VKORC1 structural homology models, the development of new effective anticoagulants has been quite complex due to the lack of structural data and reliable structure-activity relationships. However, the recent publication of VKORC1 crystal structure provides new information for further studies. Based on these findings, we combined chemical synthesis, enzymatic assays and molecular modelling methods to design a structure-activity relationship (SAR) model. Our results proved that the lipophilicity, the membrane permeability of inhibitors and their affinity towards human VKORC1 enzyme are the main characteristics for potent anticoagulants. Our SAR model managed to rank compounds according to their ability to inhibit the human VKORC1. Such a tool might constitute an alternative to evaluate new molecules potency before their chemical synthesis and biological assessment and might assist the development of new VKAs.

Structural Investigation of the Vitamin K Epoxide Reductase (VKORC1) Binding Site with Vitamin K.

The vitamin K epoxide reductase (VKORC1) enzyme is of primary importance in many physiological processes, i.e., blood coagulation, energy metabolism, and arterial calcification prevention, due to its role in the vitamin K cycle. Indeed, VKORC1 catalyzes reduction of vitamin K epoxide to quinone and then to hydroquinone. However, the three-dimensional VKORC1 structure remains experimentally undetermined, because of the endoplasmic reticulum membrane location of this enzyme. Here we present a molecular modeling investigation of the VKORC1 enzymatic site structure and function, supported by in vitro enzymatic assays. Four VKORC1 mutants were designed in silico (F55G, F55Y, N80G, and F83G) based on a previous study that identified residues F55, N80, and F83 as being crucial for vitamin K epoxide binding. F55G, N80G, and F83G nonconservative mutants were all predicted to be inactive by molecular modeling analyses. However, the F55Y conservative mutant was expected to be active compared to wild-type VKORC1. In vitro enzymatic assays performed on recombinant proteins assessed our molecular modeling hypotheses and led us to describe the role of accurate VKORC1 active site residues with respect to VKORC1. Residues F55, N80, and F83 appeared to act in a concerted manner to keep vitamin K epoxide close to the C135 catalytic residue. Residues F55 and N80 prevent naphthoquinone head rotation away from the active site, assisted by residue F83 that prevents vitamin K from sliding outside the enzymatic pocket, through hydrophobic tail stabilization. Our results thus highlighted the specific functions of VKORC1 catalytic pocket residues and evidenced the ability of our structural model to predict biological effects of VKORC1 mutations.

Comparative biological properties of the four stereoisomers of difethialone, a second-generation anticoagulant rodenticide, in rats: development of a model allowing to choose the appropriate stereoisomeric ratio.

The current management of rodent pest populations is based on second-generation anticoagulant rodenticides (SGAR). These molecules, of which difethialone is part, are much more efficient than the first generation. Nevertheless, this efficiency comes with a major drawback, SGARs are tissue persistent that increases the exposure of rodent predators to them. According to its chemical structure, difethialone has four stereoisomers, whose specific inhibition potency and pharmacokinetic have never been described and might be useful to design new eco-friendly rodenticides. The study aimed to investigate the ability to inhibit anticoagulant target enzyme (VKORC1) and the pharmacokinetics in rats of the four difethialone stereoisomers in rats. We show that stereoisomers are all highly efficient to inhibit VKORC1 activity, but they have distinct initial half-life with 6.0 h, 25.4 h, 69.3 h, and 82.3 h for, respectively, E4-trans, E2-cis, E1-trans, and E3-cis stereoisomer. These results open the way of the development of eco-friendly and efficient rodenticide by mixing some of these stereoisomers. Preferential incorporation of the E4-trans stereoisomer (high inhibitory VKORC1 potency, relatively shorter liver half-life) into difethialone rodenticides baits might result in a more eco-friendly product than current commercially available difethialone formulations. In addition, we put forward modelling to help design bait according to the circumstance of use (presence of non-target species, food competition, etc.) by modulating the theorical AUC and and the theorical concentration of the product at the death of the rodent pest. Thus, this modeling might allow to diminish the use of laboratory animal in assay.

Structural Insights into Phylloquinone (Vitamin K1), Menaquinone (MK4, MK7), and Menadione (Vitamin K3) Binding to VKORC1.

Vitamin K family molecules-phylloquinone (K1), menaquinone (K2), and menadione (K3)-act as γ-glutamyl carboxylase (GGCX)-exclusive cofactors in their hydroquinone state, activating proteins of main importance for blood coagulation in the liver and for arterial calcification prevention and energy metabolism in extrahepatic tissues. Once GGCX is activated, vitamin K is found in the epoxide state, which is then recycled to quinone and hydroquinone states by vitamin K epoxide reductase (VKORC1). Nevertheless, little information is available concerning vitamin K1, K2, or K3 tissue distribution and preferential interactions towards VKORC1. Here we present a molecular modeling study of vitamin K1, menaquinones 4, 7 (MK4, MK7), and K3 structural interactions with VKORC1. VKORC1 was shown to tightly bind vitamins K1 and MK4 in the epoxide and quinone states, but not in the hydroquinone state; five VKORC1 residues were identified as crucial for vitamin K stabilization, and two other ones were essential for hydrogen bond formation. However, vitamin MK7 revealed shaky binding towards VKORC1, induced by hydrophobic tail interactions with the membrane. Vitamin K3 exhibited the lowest affinity with VKORC1 because of the absence of a hydrophobic tail, preventing structural stabilization by the enzyme. Enzymatic activity towards vitamins K1, MK4, MK7, and K3 was also evaluated by in vitro assays, validating our in silico predictions: VKORC1 presented equivalent activities towards vitamins K1 and MK4, but much lower activity with respect to vitamin MK7, and no activity towards vitamin K3. Our results revealed VKORC1's ability to recycle both phylloquinone and some menaquinones, and also highlighted the importance of vitamin K's hydrophobic tail size and membrane interactions.

Elevated difenacoum metabolism is involved in the difenacoum-resistant phenotype observed in Berkshire rats homozygous for the L120Q mutation in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene.

BACKGROUND: Soon after difenacoum began to be used, resistance to this rodenticide was detected in rats in northeast Hampshire and northwest Berkshire in England. Resistance to difenacoum has been reported to be stronger in rats from Berkshire than in rats from Hampshire. Surprisingly, after the discovery of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene, rats from Berkshire and Hampshire were all shown to be homozygous for the L120Q mutation in Vkorc1. RESULTS: This study aimed to evaluate the resistance of Berkshire rats to confirm their extreme resistance and determine mechanisms supporting this resistance. For this purpose, we created a quasicongenic rat F7 strain by using a Berkshire rat as a donor to introduce the L120Q mutation in Vkorc1 into the genetic background of an anticoagulant-susceptible recipient strain. The use of F7 rats enabled demonstration of (i) the level of resistance to difenacoum conferred by the L120Q mutation, (ii) co-dominance of the L120 and Q120 alleles, (iii) the extreme resistance of Berkshire rats compared with Q120/Q120 rats as a consequence of additional resistance mechanisms, and (iv) the involvement of cytochrome P 450 (CYP450) enzymes in this extreme resistance. CONCLUSION: This study demonstrated that elevated CYP450 oxidative metabolism leading to accelerated difenacoum detoxification is involved in the Berkshire phenotype. © 2017 Society of Chemical Industry.

Evidence of a target resistance to antivitamin K rodenticides in the roof rat Rattus rattus: identification and characterisation of a novel Y25F mutation in the Vkorc1 gene.

BACKGROUND: In spite of intensive use of bromadiolone, rodent control was inefficient on a farm infested by rats in Zaragoza, Spain. While metabolic resistance was previously described in this rodent species, the observation of a target resistance to antivitamin K rodenticides had been poorly documented in Rattus rattus. RESULTS: From rats trapped on the farm, cytochrome b and Vkorc1 genes were amplified by PCR and sequenced in order to identify species and detect potential Vkorc1 mutations. VKORC1-deduced amino acid sequences were thus expressed in Pichia pastoris, and inhibition constants towards various rodenticides were determined. The ten rats trapped on the farm were all identified as R. rattus. They were found to be homozygous for the g.74A>T nucleotide replacement in exon 1 of the Vkorc1 gene, leading to p.Y25F mutation. This mutation led to increased apparent inhibition constants towards various rodenticides, probably caused by a partial loss of helical structure of TM4. CONCLUSION: The p.Y25F mutation detected in the Vkorc1 gene in R. rattus trapped on the Spanish farm is associated with the resistance phenotype to bromadiolone that has been observed. It is the first evidence of target resistance to antivitamin K anticoagulants in R. rattus.