Mario O. Salazar

Chemically engineered extracts: Bioactivity alteration through sulfonylation

The chemical composition and the biomolecular properties of a series of crude plant extracts were altered without previous knowledge of their detailed chemical composition.

Brominated extracts as source of bioactive compounds.

The chemical composition and the biomolecular properties of a crude plant extract were altered through bromination leading to the discovery of an acetylcholinesterase inhibitor.

Unsaturated long-chain free fatty acids are input signals of the Salmonella enterica PhoP/PhoQ regulatory system

Background: The PhoP/PhoQ system governs crucial Salmonella typhimurium pathogenic traits. Results: A screening of natural compounds showed that long chain fatty acids present in the bacterial growth medium down-regulate the PhoP/PhoQ-dependent regulon. Conclusion: Long chain unsaturated fatty acids specifically inhibit PhoQ autokinase activity. Significance: The novel PhoQ input signal reveals a new scenario for the control of Salmonella virulence, providing a rationale for future antibacterial strategies. The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg2+ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.

Discovery of a β-glucosidase inhibitor from a chemically engineered extract prepared through sulfonylation

A semisynthetic β-glucosidase inhibitor was identified from a chemically engineered extract prepared by reaction with benzenesulfonyl chloride. The structure includes a natural histamine portion and a benzenesulfonyl portion introduced during the diversification step.

Thin Layer Chromatography-Autography-High Resolution Mass Spectrometry Analysis: Accelerating the Identification of Acetylcholinesterase Inhibitors.

INTRODUCTION The prevailing treatment for Alzheimers disease is the use of acetylcholinesterase (AChE) inhibitors. Natural extracts are the principal source of AChEs inhibitors. However, their chemical complexity demands for simple, selective and rapid assays. OBJECTIVE To develop a strategy for identification of AChE inhibitors present in mixtures employing high resolution mass spectrometry (HRMS) and thin layer chromatography (TLC)-biological staining. METHODOLOGY The strategy uses an autographic assay based on the α-naphthyl acetate - fast blue B system for the detection of AChE activity. The immobilisation of AChE in agar allowed the extraction of the compounds for analysis by HRMS. Three TLC experiments employing different solvent systems were used in parallel and the mass spectra of the compounds extracted from the inhibition halos, were compared. The analysis was performed under MatLab environment. RESULTS The strategy was used to detect the presence of physostigmine in an extract of Brassica rapa L. spiked with the inhibitor. Similarly, caffeine was straightforwardly spotted as responsible for the inhibitory properties of an extract of Ilex paraguariensis Saint-Hilaire. Comparison of the HRMS profiles lead to the facile identification of the [M+H](+) and [M+Na](+) of the compounds responsible for the inhibition. CONCLUSION The proposed methodology, coupling TLC-AChE autography-HRMS, illustrates the feasibility of assigning molecular formulas of active compounds present in complex mixtures directly from autography. The new AChE agar-immobilised assay presented a more homogenous colour and a better definition than direct spraying methods, reducing the cost of the assay and improving its sensitivity.

A New Fluorinated Tyrosinase Inhibitor from a Chemically Engineered Essential Oil

The generation of fluorinated essential oils as a source of bioactive compounds is described. Most of the components of the natural mixtures were altered, leading to the discovery of a new fluorinated tyrosinase inhibitor.

A Thin-layer Chromatography Autographic Method for the Detection of Inhibitors of the Salmonella PhoP–PhoQ Regulatory System

INTRODUCTION The PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. OBJECTIVE To develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes. METHODS The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. RESULTS A bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system. CONCLUSION A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract.

Chemical diversification of essential oils, evaluation of complex mixtures and identification of a xanthine oxidase inhibitor

A set of chemically engineered essential oils has been generated through chemical diversification by reaction with bromine. The impact of the reaction over the chemical composition of the mixtures was qualitatively demonstrated through GC-MS and utilizing multivariate analysis of 1H NMR and GC-MS. Most of the components of the essential oils are transformed by the reaction expanding the chemical diversity of the mixtures. Biological changes between essential oils and brominated essential oils were demonstrated through image analysis of xanthine oxidase autography profiles. The highest biological activity increase was obtained for the Foeniculum vulgare Mill essential oil. Coupling of xanthine oxidase autography with the BIOMSID strategy allowed the identification of the molecular formula of the active compound. Bioguided fractionation of the mixture led to the isolation of (RS)-2-bromo-1-(4-methoxyphenyl) propan-1-one for being responsible for the observed bioactivity. This xanthine oxidase inhibitor could have been formed from the inactive natural component anethole. The inhibitory potency of this semisynthetic compound was in the same order of magnitude as allopurinol, the most used inhibitor.

N α -arylsulfonyl histamines as selective β-glucosidase inhibitors

N α -benzenesulfonylhistamine, a new semi-synthetic β-glucosidase inhibitor, was obtained by bioactivity-guided isolation from a chemically engineered extract of Urtica urens L. prepared by reaction with benzenesulfonyl chloride. In order to identify better β-glucosidase inhibitors, a new series of Nα,Nτ-di-arylsulfonyl and Nα-arylsulfonyl histamine derivatives was prepared. Biological studies revealed that the β-glucosidase inhibition was in a micromolar range for several Nα-arylsulfonyl histamine compounds of the series, Nα-4-fluorobenzenesulfonyl histamine being the most powerful compound. Besides, this reversible and competitive inhibitor presented a good selectivity for β-glucosidase with respect to other target enzymes including α-glucosidase.