Promising antibacterial efficacy of arenicin peptides against the emerging opportunistic pathogen Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus, a fast-growing non-tuberculous mycobacterium, is an emerging opportunistic pathogen responsible for chronic bronchopulmonary infections in people with respiratory diseases such as cystic fibrosis (CF). Due to its intrinsic polyresistance to a wide range of antibiotics, most treatments for M. abscessus pulmonary infections are poorly effective. In this context, antimicrobial peptides (AMPs) active against bacterial strains and less prompt to cause resistance, represent a good alternative to conventional antibiotics. Herein, we evaluated the effect of three arenicin isoforms, possessing two or four Cysteines involved in one (Ar-1, Ar-2) or two disulfide bonds (Ar-3), on the in vitro growth of M. abscessus. METHODS: The respective disulfide-free AMPs, were built by replacing the Cysteines with alpha-amino-n-butyric acid (Abu) residue. We evaluated the efficiency of the eight arenicin derivatives through their antimicrobial activity against M. abscessus strains, their cytotoxicity towards human cell lines, and their hemolytic activity on human erythrocytes. The mechanism of action of the Ar-1 peptide was further investigated through membrane permeabilization assay, electron microscopy, lipid insertion assay via surface pressure measurement, and the induction of resistance assay. RESULTS: Our results demonstrated that Ar-1 was the safest peptide with no toxicity towards human cells and no hemolytic activity, and the most active against M. abscessus growth. Ar-1 acts by insertion into mycobacterial lipids, resulting in a rapid membranolytic effect that kills M. abscessus without induction of resistance. CONCLUSION: Overall, the present study emphasized Ar-1 as a potential new alternative to conventional antibiotics in the treatment of CF-associated bacterial infection related to M. abscessus.

Evaluation of the Efficiency of Random and Diblock Methacrylate-Based Amphiphilic Cationic Polymers against Major Bacterial Pathogens Associated with Cystic Fibrosis.

Cystic fibrosis (CF) is associated with repeated lung bacterial infection, mainly by Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium abscessus, all known to be or becoming resistant to several antibiotics, often leading to therapeutic failure and death. In this context, antimicrobial peptides and antimicrobial polymers active against resistant strains and less prompt to cause resistance, appear as a good alternative to conventional antibiotics. In the present study, methacrylate-based copolymers obtained by radical chemistry were evaluated against CF-associated bacterial strains. Results showed that the type (Random versus Diblock) and the size of the copolymers affected their antibacterial activity and toxicity. Among the different copolymers tested, four (i.e., Random10200, Random15000, Random23900, and Diblock9500) were identified as the most active and the safest molecules and were further investigated. Data showed that they inserted into bacterial lipids, leading to a rapid membranolytic effect and killing of the bacterial. In relation with their fast bactericidal action and conversely to conventional antibiotics, those copolymers did not induce a resistance and remained active against antibiotic-resistant strains. Finally, the selected copolymers possessed a preventive effect on biofilm formation, although not exhibiting disruptive activity. Overall, the present study demonstrates that methacrylate-based copolymers are an interesting alternative to conventional antibiotics in the treatment of CF-associated bacterial infection.

Direct capture, inhibition and crystal structure of HsaD (Rv3569c) from M. tuberculosis.

A hallmark of Mycobacterium tuberculosis (M. tb), the aetiologic agent of tuberculosis, is its ability to metabolise host-derived lipids. However, the enzymes and mechanisms underlying such metabolism are still largely unknown. We previously reported that the Cyclophostin & Cyclipostins (CyC) analogues, a new family of potent antimycobacterial molecules, react specifically and covalently with (Ser/Cys)-based enzymes mostly involved in bacterial lipid metabolism. Here, we report the synthesis of new CyC alkyne-containing inhibitors (CyCyne ) and their use for the direct fishing of target proteins in M. tb culture via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the capture and identification of a variety of enzymes, and many of them involved in lipid or steroid metabolisms. One of the captured enzymes, HsaD (Rv3569c), is required for the survival of M. tb within macrophages and is thus a potential therapeutic target. This prompted us to further explore and validate, through a combination of biochemical and structural approaches, the specificity of HsaD inhibition by the CyC analogues. We confirmed that the CyC bind covalently to the catalytic Ser114 residue, leading to a total loss of enzyme activity. These data were supported by the X-ray structures of four HsaD-CyC complexes, obtained at resolutions between 1.6 and 2.6 Å. The identification of mycobacterial enzymes directly captured by the CyCyne probes through CC-ABPP paves the way to better understand and potentially target key players at crucial stages of the bacilli life cycle.

Early life oxytocin treatment improves thermo-sensory reactivity and maternal behavior in neonates lacking the autism-associated gene Magel2.

Atypical responses to sensory stimuli are considered as a core aspect and early life marker of autism spectrum disorders (ASD). Although recent findings performed in mouse ASD genetic models report sensory deficits, these were explored exclusively during juvenile or adult period. Whether sensory dysfunctions might be present at the early life stage and rescued by therapeutic strategy are fairly uninvestigated. Here we found that under cool environment neonatal mice lacking the autism-associated gene Magel2 present pup calls hypo-reactivity and are retrieved with delay by their wild-type dam. This neonatal atypical sensory reactivity to cool stimuli was not associated with autonomic thermoregulatory alteration but with a deficit of the oxytocinergic system. Indeed, we show in control neonates that pharmacogenetic inactivation of hypothalamic oxytocin neurons mimicked atypical thermosensory reactivity found in Magel2 mutants. Furthermore, pharmacological intranasal administration of oxytocin to Magel2 neonates was able to rescue both the atypical thermosensory response and the maternal pup retrieval. This preclinical study establishes for the first-time early life impairments in thermosensory integration and suggest a therapeutic potential benefit of intranasal oxytocin treatment on neonatal atypical sensory reactivity for autism.

Intrabacterial lipid inclusions in mycobacteria: unexpected key players in survival and pathogenesis?

Mycobacterial species, including Mycobacterium tuberculosis, rely on lipids to survive and chronically persist within their hosts. Upon infection, opportunistic and strict pathogenic mycobacteria exploit metabolic pathways to import and process host-derived free fatty acids, subsequently stored as triacylglycerols in the form of intrabacterial lipid inclusions (ILI). Under nutrient-limiting conditions, ILI constitute a critical source of energy that fuels the carbon requirements and maintain redox homeostasis, promoting bacterial survival for extensive periods of time. In addition to their basic metabolic functions, these organelles display multiple other biological properties, emphasizing their central role in the mycobacterial life cycle. However, despite their importance, the dynamics of ILI metabolism and their contribution to mycobacterial adaptation/survival in the context of infection has not been thoroughly documented. Herein, we provide an overview of the historical ILI discoveries, their characterization and current knowledge regarding the microenvironmental stimuli conveying ILI formation, storage and degradation. We also review new biological systems to monitor the dynamics of ILI metabolism in extra- and intracellular mycobacteria and describe major molecular actors in triacylglycerol biosynthesis, maintenance and breakdown. Finally, emerging concepts regarding the role of ILI in mycobacterial survival, persistence, reactivation, antibiotic susceptibility and inter-individual transmission are also discussed.

Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria.

Mycobacteria share with other actinomycetes the ability to produce large quantities of triacylglycerol (TAG), which accumulate as intracytoplasmic lipid inclusions (ILI) also known as lipid droplets (LD). Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis, acquires fatty acids from the human host which are utilized to synthesize TAG, subsequently stored in the form of ILI to meet the carbon and nutrient requirements of the bacterium during long periods of persistence. However, environmental factors governing mycobacterial ILI formation and degradation remain poorly understood. Herein, we demonstrated that in the absence of host cells, carbon excess and nitrogen starvation promote TAG accumulation in the form of ILI in M. smegmatis and M. abscessus, used as surrogate species of M. tb. Based on these findings, we developed a simple and reversible in vitro model to regulate ILI biosynthesis and hydrolysis in mycobacteria. We also showed that TAG formation is tgs1 dependent and that lipolytic enzymes mediate TAG breakdown. Moreover, we confirmed that the nitrogen-deprived and ILI-rich phenotype was associated with an increased tolerance towards several drugs used for treating mycobacterial infections. Importantly, we showed that the presence of ILI substantially enhanced the bacterial burden and granuloma abundance in zebrafish embryos infected with lipid-rich M. abscessus as compared to embryos infected with lipid-poor M. abscessus, suggesting that ILI are actively contributing to mycobacterial virulence and pathogenesis.

Dissecting the membrane lipid binding properties and lipase activity of Mycobacterium tuberculosis LipY domains.

The Mycobacterium tuberculosis LipY protein, a prototype of the proline-glutamic acid (PE) family, exhibits a triacylglycerol (TAG) hydrolase activity that contributes to host cell lipid degradation and persistence of the bacilli. LipY is found either as a full-length intracytosolic form or as a mature extracellular form lacking the N-terminal PE domain. Even though the contribution of the extracellular form in TAG consumption has been partly elucidated, very little information is available regarding the potential interactions of either full-length LipY with the cytoplasmic membrane, or mature form LipY with the outer membrane. Herein, several LipY variants truncated in their N-terminal domain were produced and biochemically characterized in lipid-protein interaction assays, using the monomolecular film technique and FTIR. Comparison of the catalytic activities of these recombinant proteins showed that LipY∆149, corresponding to the extracellular form of LipY lacking the PE domain, is more active than the full-length protein. This confirms previous studies reporting that the PE domain negatively modulates the TAG hydrolase activity of LipY. Lipid-protein interaction studies indicate that the PE domain anchors LipY onto membrane lipids. Consistent with these findings, we show that LipY∆149 is loosely associated with the mycobacterial cell wall, and that this interaction is mediated by the sole lipase domain. Overall, our results bring new information regarding the molecular mechanisms by which LipY either binds and hydrolyses host cell lipids or degrades TAG, the major source of lipids within mycobacterial intracytosolic lipid inclusions.

Haloarcula sebkhae sp. nov., an extremely halophilic archaeon from Algerian hypersaline environment.

A halophilic organism, SWO25T, was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25T grew optimally at 35-45 °C, at pH 6.0-8.0 and 0.05-0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3-5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25T to the genus Haloarcula. The DNA G+C content was 61.1mol%. DNA-DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB' genes showed that strain SWO25T is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarculasebkhae sp. nov. is proposed. The type strain is SWO25T (=CIP 110583T=JCM 19018T).

LipG a bifunctional phospholipase/thioesterase involved in mycobacterial envelope remodeling.

Tuberculosis caused by Mycobacterium tuberculosis is currently one of the leading causes of death from an infectious agent. The main difficulties encountered in eradicating this bacteria are mainly related to (i) a very complex lipid composition of the bacillus cell wall, (ii) its ability to hide from the immune system inside the granulomas, and (iii) the increasing number of resistant strains. In this context, we were interested in the Rv0646c (lipGMTB ) gene located upstream to the mmaA cluster which is described as being crucial for the production of cell wall components and required for the bacilli adaptation and survival in mouse macrophages. Using biochemical experiments combined with the construction of deletion and overexpression mutant strains in Mycobacterium smegmatis, we found that LipGMTB is a cytoplasmic membrane-associated enzyme that displays both phospholipase and thioesterase activities. Overproduction of LipGMTB decreases the glycopeptidolipids (GPL) level concomitantly to an increase in phosphatidylinositol (PI) which is the precursor of the PI mannoside (PIM), an essential lipid component of the bacterial cell wall. Conversely, deletion of the lipGMS gene in M. smegmatis leads to an overproduction of GPL, and subsequently decreases the strain susceptibility to various antibiotics. All these findings demonstrate that LipG is involved in cell envelope biosynthesis/remodeling, and consequently this enzyme may thus play an important role in mycobacterial physiology.

Slowing down fat digestion and absorption by an oxadiazolone inhibitor targeting selectively gastric lipolysis.

Based on a previous study and in silico molecular docking experiments, we have designed and synthesized a new series of ten 5-Alkoxy-N-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one derivatives (RmPPOX). These molecules were further evaluated as selective and potent inhibitors of mammalian digestive lipases: purified dog gastric lipase (DGL) and guinea pig pancreatic lipase related protein 2 (GPLRP2), as well as porcine (PPL) and human (HPL) pancreatic lipases contained in porcine pancreatic extracts (PPE) and human pancreatic juices (HPJ), respectively. These compounds were found to strongly discriminate classical pancreatic lipases (poorly inhibited) from gastric lipase (fully inhibited). Among them, the 5-(2-(Benzyloxy)ethoxy)-3-(3-PhenoxyPhenyl)-1,3,4-Oxadiazol-2(3H)-one (BemPPOX) was identified as the most potent inhibitor of DGL, even more active than the FDA-approved drug Orlistat. BemPPOX and Orlistat were further compared in vitro in the course of test meal digestion, and in vivo with a mesenteric lymph duct cannulated rat model to evaluate their respective impacts on fat absorption. While Orlistat inhibited both gastric and duodenal lipolysis and drastically reduced fat absorption in rats, BemPPOX showed a specific action on gastric lipolysis that slowed down the overall lipolysis process and led to a subsequent reduction of around 55% of the intestinal absorption of fatty acids compared to controls. All these data promote BemPPOX as a potent candidate to efficiently regulate the gastrointestinal lipolysis, and to investigate its link with satiety mechanisms and therefore develop new strategies to "fight against obesity".

New lipase assay using Pomegranate oil coating in microtiter plates.

Lipases play various roles in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid bodies in animals, plants and microorganisms. In association with these physiological functions, there is an important field of research for discovering lipase inhibitors and developing new treatments of diseases such as obesity, atherosclerosis, diabetes and tuberculosis. In this context, the development of convenient, specific and sensitive analytical methods for the detection and assay of lipases and/or lipase inhibitors is of major importance. It is shown here that purified triacylglycerols (TAGs) from Punica granatum (Pomegranate) seed oil coated on microtiter plates can be used for the continuous assay of lipase activity by recording the variations with time of the UV absorption spectra at 275 nm. UV absorption is due the release of punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid), a conjugated triene contained in Pomegranate oil. This new microtiter plate assay allows to accurately measure the activity of a wider range of lipases compared to the similar assay previously developed with Tung oil containing α-eleostearic acid (9Z,11E,13E-octadeca-9,11,13-trienoic acid), including the LipY lipase from Mycobacterium tuberculosis. Although punicic acid is a diastereoisomer of α-eleostearic acid, the Δ(13)cis double bound found in punicic acid gives a different structure to the acyl chain that probably favours the interaction of Pomegranate TAGs with the lipase active site. The microplate lipase assay using Pomegranate TAGs shows high sensitivity, reproducibility and remarkable relevance for the high-speed screening of lipases and/or lipase inhibitors directly from raw culture media without any purification step.

Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.

In vitro digestion of citric acid esters of mono- and diglycerides (CITREM) and CITREM-containing infant formula/emulsions.

CITREM is an emulsifier used in the food industry and contains citric acid esters of mono- and diglycerides (GCFE). It is generally recognized as safe but no publication on its digestibility under gastrointestinal conditions and impact on fat digestion was available. It was shown here that fatty acids are released from CITREM by gastric lipase, pancreatic lipase, pancreatic-lipase-related protein 2 and carboxyl ester hydrolase. A two-step in vitro digestion model mimicking lipolysis in the stomach and upper small intestine of term and preterm infants was then used to evaluate the digestibility of CITREM alone, CITREM-containing infant formula and fat emulsions, and isolated GCFE fractions. Overall, it was shown that fat digestion is not significantly changed by the presence of CITREM, and fatty acids contained in CITREM compounds are released to a large extent by lipases. Nevertheless, undigestible water-soluble compounds containing glycerol and citric acid units were identified, indicating that the ester bond between citric acid and glycerol is not fully hydrolyzed throughout the proposed digestion.

An interfacial and comparative in vitro study of gastrointestinal lipases and Yarrowia lipolytica LIP2 lipase, a candidate for enzyme replacement therapy.

Lipolytic activities of Yarrowia lipolytica LIP2 lipase (YLLIP2), human pancreatic (HPL) and dog gastric (DGL) lipases were first compared using lecithin-stabilized triacylglycerol (TAG) emulsions (Intralipid) at various pH and bile salt concentrations. Like DGL, YLLIP2 was able to hydrolyze TAG droplets covered by a lecithin monolayer, while HPL was not directly active on that substrate. These results were in good agreement with the respective kinetics of adsorption on phosphatidylcholine (PC) monomolecular films of the same three lipases, YLLIP2 being the most tensioactive lipase. YLLIP2 adsorption onto a PC monolayer spread at the air/water interface was influenced by pH-dependent changes in the enzyme/lipid interfacial association constant (KAds) which was optimum at pH 6.0 on long-chain egg PC monolayer, and at pH 5.0 on medium chain dilauroylphosphatidylcholine film. Using substrate monolayers (1,2-dicaprin, trioctanoin), YLLIP2 displayed the highest lipolytic activities on both substrates in the 25-35 mN m(-1) surface pressure range. YLLIP2 was active in a large pH range and displayed a pH-dependent activity profile combining DGL and HPL features at pH values found in the stomach (pH 3-5) and in the intestine (pH 6-7), respectively. The apparent maximum activity of YLLIP2 was observed at acidic pH 4-6 and was therefore well correlated with an efficient interfacial binding at these pH levels, whatever the type of interfaces (Intralipid emulsions, substrate or PC monolayers). All these findings support the use of YLLIP2 in enzyme replacement therapy for the treatment of pancreatic exocrine insufficiency, a pathological situation in which an acidification of intestinal contents occurs.

Using the reversible inhibition of gastric lipase by Orlistat for investigating simultaneously lipase adsorption and substrate hydrolysis at the lipid-water interface.

The lipolysis reaction carried out by lipases at the water-lipid interface is a complex process including enzyme conformational changes, adsorption/desorption equilibrium and substrate hydrolysis. Mixed monomolecular films of the lipase inhibitor Orlistat and 1,2-dicaprin were used here to investigate the adsorption of dog gastric lipase (DGL) followed by the hydrolysis of 1,2-dicaprin. The combined study of these two essential catalysis steps was made possible thanks to the highest affinity of DGL for Orlistat than 1,2-dicaprin and the fact that the inhibition of DGL by Orlistat is reversible. Upon DGL binding to mixed 1,2-dicaprin/Orlistat monolayers, an increase in surface pressure reflecting lipase adsorption was first recorded. Limited amounts of Orlistat allowed to maintain DGL inactive on 1,2-dicaprin during a period of time that was sufficient to determine DGL adsorption and desorption rate constants. A decrease in surface pressure reflecting 1,2-dicaprin hydrolysis and product desorption was observed after the slow hydrolysis of the covalent DGL-Orlistat complex was complete. The rate of 1,2-dicaprin hydrolysis was recorded using the surface barostat technique. Based on a kinetic model describing the inhibition by Orlistat and the activity of DGL on a mixed 1,2-dicaprin/Orlistat monolayer spread at the air-water interface combined with surface pressure measurements, it was possible to monitor DGL adsorption at the lipid-water interface and substrate hydrolysis in the course of a single experiment. This allowed to assess the kcat/KM* ratio for DGL acting on 1,2-dicaprin monolayer, after showing that mixed monolayers containing a low fraction of Orlistat were similar to pure 1,2-dicaprin monolayers.

New insights into the pH-dependent interfacial adsorption of dog gastric lipase using the monolayer technique.

The access to kinetic parameters of lipolytic enzyme adsorption onto lipids is essential for a better understanding of interfacial enzymology and lipase-lipid interactions. The interfacial adsorption of dog gastric lipase (DGL) was monitored as a function of pH and surface pressure (Π), independently from the catalytic activity, using non-hydrolysable 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) monomolecular films. The acid-stable DGL, which initiates fat digestion in the stomach, was then selected because its adsorption kinetics onto hydrophobic solid surfaces were already studied. This gastric lipase was therefore used as a model enzyme to validate both experimental and theoretical approaches. Results show that the adsorption process of DGL at the lipid/water interface depends on a pH-dependent adsorption equilibrium coefficient which is optimum at pH 5.0 (K(Ads) = 1.7 ± 0.05 × 10(8)M(-1)). KAds values further allowed an indirect estimation of the molar fraction (ΦE*(%), mol%) as well as the molecular area (AE*) of DGL adsorbed onto DLPC monolayer. Based on these data, a model for DGL adsorption onto DLPC monolayer at pH 5.0 is proposed for a surface pressure range of 15-25 mNm(-1).

Enantioselective inhibition of microbial lipolytic enzymes by nonracemic monocyclic enolphosphonate analogues of cyclophostin.

Four nonracemic enolphosphonate analogues of Cyclophostin were obtained by asymmetric synthesis, and their absolute configurations at both phosphorus and C-5 carbon chiral centers were unambiguously assigned. The influence of chirality was studied by testing the inhibitory effects of these four stereoisomers toward the lipolytic activity of three microbial lipases: Fusarium solani cutinase, Rv0183, and LipY from Mycobacterium tuberculosis . Cutinase was highly diastereoselective for the (Sp) configuration using (Sc) inhibitors, whereas no obvious stereopreference at phosphorus was observed with (Rc) compounds. Conversely, Rv0183 exhibited strong enantioselective discrimination for (Sp) configuration regardless of the chirality at the asymmetric carbon atom. Lastly, LipY discriminated only the unusual diastereoisomeric configuration (Rc, Rp) leading to the most potent inhibitor. This work, which provides a fundamental premise for the understanding of the stereoselective relationships between nonracemic enolphosphonates and their inhibitory activity, also opens new prospects on the design and synthesis of highly specific enantioselective antimicrobial agents.

Effects of the propeptide of group X secreted phospholipase A(2) on substrate specificity and interfacial activity on phospholipid monolayers.

Group X secreted phospholipase A(2) (GX sPLA(2)) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA(2) activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA(2) activity of recombinant mouse GX sPLA(2) (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA(2) activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30-35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA(2) activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA(2). This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance.

Analysis of the discriminative inhibition of mammalian digestive lipases by 3-phenyl substituted 1,3,4-oxadiazol-2(3H)-ones.

We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e.MmPPOX and MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human (HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipase-related proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts (PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of DGL, we demonstrated that the phenoxy group plays a key role in specific molecular interactions within the lipase's active site. The absence of this group in the case of MPOX, as well as its connectivity to the neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory efficiency of these oxadiazolones and leads to a significant gain in selectivity towards the lipases tested. The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone derivatives could also provide useful leads for the development of novel and more discriminative inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal lipolysis process.

Synthesis and kinetic evaluation of cyclophostin and cyclipostins phosphonate analogs as selective and potent inhibitors of microbial lipases.

A new series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. The best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents.