Mohamed Haddad

NL MIND-BEST : a web server for ligands and proteins discovery--theoretic-experimental study of proteins of Giardia lamblia and new compounds active against Plasmodium falciparum

There are many protein ligands and/or drugs described with very different affinity to a large number of target proteins or receptors. In this work, we selected Ligands or Drug-target pairs (DTPs/nDTPs) of drugs with high affinity/non-affinity for different targets. Quantitative Structure-Activity Relationships (QSAR) models become a very useful tool in this context to substantially reduce time and resources consuming experiments. Unfortunately most QSAR models predict activity against only one protein target and/or have not been implemented in the form of public web server freely accessible online to the scientific community. To solve this problem, we developed here a multi-target QSAR (mt-QSAR) classifier using the MARCH-INSIDE technique to calculate structural parameters of drug and target plus one Artificial Neuronal Network (ANN) to seek the model. The best ANN model found is a Multi-Layer Perceptron (MLP) with profile MLP 20:20-15-1:1. This MLP classifies correctly 611 out of 678 DTPs (sensitivity=90.12%) and 3083 out of 3408 nDTPs (specificity=90.46%), corresponding to training accuracy=90.41%. The validation of the model was carried out by means of external predicting series. The model classifies correctly 310 out of 338 DTPs (sensitivity=91.72%) and 1527 out of 1674 nDTP (specificity=91.22%) in validation series, corresponding to total accuracy=91.30% for validation series (predictability). This model favorably compares with other ANN models developed in this work and Machine Learning classifiers published before to address the same problem in different aspects. We implemented the present model at web portal Bio-AIMS in the form of an online server called: Non-Linear MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (NL MIND-BEST), which is located at URL: http://miaja.tic.udc.es/Bio-AIMS/NL-MIND-BEST.php. This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally we illustrated two practical uses of this server with two different experiments. In experiment 1, we report by first time Quantum QSAR study, synthesis, characterization, and experimental assay of antiplasmodial and cytotoxic activities of oxoisoaporphine alkaloids derivatives as well as NL MIND-BEST prediction of potential target proteins. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF, and -TOF/TOF MS, MASCOT search, MM/MD 3D structure modeling, and NL MIND-BEST prediction for different peptides a new protein of the found in the proteome of the human parasite Giardia lamblia, which is promising for anti-parasite drug-targets discovery.

Protective effect of Heliotropium foertherianum (Boraginaceae) folk remedy and its active compound, rosmarinic acid, against a Pacific ciguatoxin.

ETHNOPHARMACOLOGICAL RELEVANCE Senescent leaves of Heliotropium foertherianum Diane & Hilger (Boraginaceae) are traditionally used in the Pacific region to treat Ciguatera Fish Poisoning. This plant contains rosmarinic acid that is known for its multiple biological activities. In the present study, H. foertherianum aqueous extract, rosmarinic acid and its derivatives were evaluated for their capacity to reduce the effect of ciguatoxins. MATERIALS AND METHODS Aqueous extract of H. foertherianum leaves was prepared and studied for its effects against a Pacific ciguatoxin (P-CTX-1B) in the neuroblastoma cell assay and the receptor binding assay. Rosmarinic acid and six derivatives were also evaluated by means of these bioassays. For this purpose, we have developed an improved synthetic route for caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE). RESULTS Both the aqueous extract of H. foertherianum leaves and rosmarinic acid showed inhibitory activities against a Pacific ciguatoxin in the above bioassays. Among all the molecules that were evaluated, rosmarinic acid was the most active compound. CONCLUSION These results confirm further the potential of H. foertherianum in the treatment of Ciguatera Fish Poisoning.

A review of traditional remedies of ciguatera fish poisoning in the Pacific.

Ciguatera fish poisoning (CFP) is an illness caused by eating tropical coral fish contaminated with ciguatoxins (CTXs). The clinical management of patients with CFP is generally supportive and symptomatic in nature as no antidote exists. Of the many drugs prescribed, several have been claimed to be efficient in small, uncontrolled studies, but the outcomes of treatments with these medicines are often contradictory. In New Caledonia, traditional remedies are commonly employed in the treatment of CFP and of the 90 plant species catalogued as useful in CFP, the most popular herbal remedy by far is a decoction prepared from the leaves of Heliotropium foertherianum Diane & Hilger (Boraginaceae). Other important plants used in the treatment of CFP include Euphorbia hirta L. (Euphorbiaceae) and Vitex L. sp. (Lamiaceae). This review focuses on the evidence for efficacy of these species and pharmacological studies which support their use. Other plants used in CFP and the conventional treatment of CFP are also discussed briefly. Copyright

Evaluation of antiplasmodial and antileishmanial activities of herbal medicine Pseudelephantopus spiralis (Less.) Cronquist and isolated hirsutinolide-type sesquiterpenoids

ETHNOPHARMACOLOGICAL RELEVANCE Pseudelephantopus spiralis (Less.) Cronquist is distributed in the Caribbean, Mesoamerica and Latin America. Preparations of the plant are traditionally used in Latin America for the treatment of various diseases including fever, malaria, and spleen or liver inflammations. MATERIALS AND METHODS Aerial parts of P. spiralis were extracted with either ethanol or distilled water. Seven hirsutinolide-type sesquiterpenoids were isolated: 8-acetyl-13-ethoxypiptocarphol (1), diacetylpiptocarphol (2), piptocarphins A (3), F (4) and D (5), (1S(*),4R(*),8S(*),10R(*))-1,4-epoxy-13-ethoxy-1,8,10-trihydroxygermacra-5E,7(11)-dien-6,12-olide (6), and piptocarphol (7). Extracts and isolated compounds (2, 3, 5-7) were screened for their in vitro antiplasmodial activity against the chloroquine-resistant Plasmodium falciparum strain FcM29-Cameroon and antileishmanial activity against three stages of Leishmania infantum. Their cytotoxicities were also evaluated against healthy VERO cell lines and J774A.1 macrophages, the host cells of the Leishmania parasites in humans. RESULTS Aqueous extracts showed a greater inhibitory effect than alcoholic extracts, with IC50 on P. falciparum of 3.0µg/mL versus 21.1µg/mL, and on L. infantum of 13.4µg/mL versus >50µg/mL. Both extracts were found to be cytotoxic to VERO cells (CC50<3µg/mL). Sesquiterpene lactones 2 and 3 showed the best activity against both parasites but failed in selectivity. Carbon 8 hydroxylated hirsutinolides 5-7 presented the particularity of exhibiting two conformers observed in solution during extensive NMR analyses in CD3OD and UHPLC-MS. The presence of a hydroxyl function at C-8 decreased the activity of 5-7 on the two parasites and also on VERO cells. CONCLUSION The antiplasmodial activity displayed by the aqueous extract explains the traditional use of P. spiralis in the treatment of malaria. This activity seems to be attributable to the presence of sesquiterpene lactones 2 and 3, the most active against P. falciparum. Aqueous extract and compounds 2, 3 and 6 were also active against L. infantum but lacked in selectivity due to their cytotoxicity towards macrophages. Exploring the safety and antiplasmodial efficacy of this traditional remedy will require further toxicological and in vivo studies in the light of the cytotoxicity towards healthy cell lines displayed by the aqueous extract and compounds 2 and 3.

Impact of metal stress on the production of secondary metabolites in Pteris vittata L. and associated rhizosphere bacterial communities

Plants adapt to metal stress by modifying their metabolism including the production of secondary metabolites in plant tissues. Such changes may impact the diversity and functions of plant associated microbial communities. Our study aimed to evaluate the influence of metals on the secondary metabolism of plants and the indirect impact on rhizosphere bacterial communities. We then compared the secondary metabolites of the hyperaccumulator Pteris vittata L. collected from a contaminated mining site to a non-contaminated site in Vietnam and identified the discriminant metabolites. Our data showed a significant increase in chlorogenic acid derivatives and A-type procyanidin in plant roots at the contaminated site. We hypothesized that the intensive production of these compounds could be part of the antioxidant defense mechanism in response to metals. In parallel, the structure and diversity of bulk soil and rhizosphere communities was studied using high-throughput sequencing. The results showed strong differences in bacterial composition, characterized by the dominance of Proteobacteria and Nitrospira in the contaminated bulk soil, and the enrichment of some potential human pathogens, i.e., Acinetobacter, Mycobacterium, and Cupriavidus in P. vittata’s rhizosphere at the mining site. Overall, metal pollution modified the production of P. vittata secondary metabolites and altered the diversity and structure of bacterial communities. Further investigations are needed to understand whether the plant recruits specific bacteria to adapt to metal stress.

Analysis and fragmentation mechanisms of hirsutinolide‐type sesquiterpene lactones by ultra‐high‐performance liquid chromatography/electrospray ionization linear ion trap Orbitrap mass spectrometry

RATIONALE Hirsutinolide-type sesquiterpene lactones (SLs) are natural biologically active compounds mainly found in the genus Vernonia. Very few studies have been published about the fragmentation mechanisms of SLs generally and none about hirsutinolides, although they have drawn attention through their biological and taxonomical interest. This work aims to propose a mass spectrometry fragmentation pattern for hirsutinolides in order to detect and to identify them in a botanical extract. METHODS The fragmentation pathways of six pure hirsutinolides isolated from Pseudelephantopus spiralis were established by positive ion electrospray high-resolution linear ion trap Orbitrap tandem mass spectrometry (ESI(+)-HRMS(n) ). A resolutive, hyphenated ultra-high-performance liquid chromatography (UHPLC) coupled to diode array detection (DAD) and ESI(+)-HRMS(n) method was then implemented to separate and analyze them. The ionization behaviour and diagnostic product ions were investigated by both methods. The UHPLC/DAD-ESI-HRMS(n) method was applied for the dereplication of a plant extract. RESULTS For the six standard compounds, the main fragmentation pattern consists first in the loss of the side chain in the C-8 position followed by the loss of the substituent in the C-13 position. UHPLC/HRMS analyses of hirsutinolides mainly produced sodiated molecules or [M+H-H2 O](+) ions. The high-abundance product ions at m/z 299 and 259 were established to be the characteristic diagnostic ions of the hirsutinolide core. The analysis of a P. spiralis extract further led to the identification of two putative hirsutinolides. CONCLUSIONS The UHPLC/DAD-HRMS(n) method combining characteristic fragmentation patterns and the profiles of the product ions generated in the MS and MS/MS spectra is an effective technique for characterizing hirsutinolide-type SLs.

Development and Validation of Liquid Chromatography Combined with Tandem Mass Spectrometry Methods for the Quantitation of Simalikalactone E in Extracts of Quassia amara L. and in Mouse Blood

INTRODUCTION Simalikalactone E (SkE) from Quassia amara, has been proved to be a valuable anti-malarial and anti-cancer compound. As SkE is very scarce, methods of quantitation are needed in order to optimise its isolation process and to determine pharmacokinetic data. OBJECTIVE To validate methods using liquid chromatography coupled to mass spectrometry for the quantitation of SkE in plant extracts and in biological fluids. METHODS High- and ultrahigh-performance liquid chromatography (UHPLC) coupled to ion trap mass spectrometry (MS) with single ion monitoring detection and to triple quadrupole-linear ion trap tandem mass spectrometry with multiple reaction monitoring detection methods were developed. Validation procedure was realised according to the International Conference on Harmonisation guideline. Methanol extracts of dried Quassia amara leaves, and mouse-blood samples obtained after various routes of administration, were analysed for SkE. RESULTS Methods were validated and gave similar results regarding the content of SkE expressed per kilogram of dry leaves in the traditional decoction (160 ± 12 mg/kg) and in the methanol extract (93 ± 2 mg/kg). The recovery of the analyte from mouse blood ranged from 80.7 to 119.8%. Simalikalactone E was only detected using UHPLC-MS/MS (0.2 ± 0.03 mg/L) in mouse blood after intravenous injection: none was detected following intraperitoneal or oral gavage administration of SkE. CONCLUSION The LC-MS methods were used for the quantitation of SkE in plant extracts and in mouse blood. These methods open the way for further protocol optimisation of SkE extraction and the determination of its pharmacokinetic data.

Adaptation of a microbead assay for the easy evaluation of traditional anti-sickling medicines: application to DREPANOSTAT and FACA

Abstract Context: Sickle cell disease is a common inherited blood disorder affecting millions of people worldwide. Due to lack of progress in drug discovery for a suitable treatment, sufferers often turn to traditional medicines that take advantage of the plant extracts activity used by traditional healers. Objective: This study optimizes an anti-sickling screening test to identify preparations capable of reverting sickle cells back to the morphology of normal red blood cells. We focused on the miniaturization and practicability of the assay, so that it can be adapted to the laboratory conditions commonly found in less developed countries. Materials and methods: We tested two traditional anti-sickling herbal medicines, FACA® and DREPANOSTAT®, composed of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Rutaceae) and Calotropis procera (Aiton) Dryand. (Apocynaceae) at screening concentrations of hydroethanol extracts from 0.2 to 1 mg/mL. Potential bioactive molecules present in the extracts were profiled using Ultra High Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UHPLC-HRMS/MS) method, identified through HRMS, MS/MS spectra and in silico fragmentation tools. Results: Hydroethanol extracts of FACA® and DREPANOSTAT® showed low anti-sickling activity, inhibiting less than 10% of the sickling process. The UHPLC-HRMS/MS profiles identified 28 compounds (18 in FACA® and 15 in DREPANOSTAT®, including common compounds) among which l-phenylalanine is already described as potential anti-sickling agent. When used as positive control, 7 mg/mL phenylalanine reduced the sickled RBC to 52%. Discussion and conclusions: This assay has been optimized for the easy screening of plant extracts or extracted compounds from bioassay guided fractionation, valuable to laboratories from less developed countries.

Natural Products as Antiparasitic Agents

Parasitic diseases remain a major burden on global human and veterinary health. They affect more than two billion people worldwide causing considerable morbidity and mortality and are a major constraint on livestock production, especially in the world’s poorest communities. The immense suffering caused by these illnesses and the consequential loss of productivity is a major drain on the limited resources of the populations in which they occur. Most modern and effective drugs for parasitic diseases present no financial viability for the pharmaceutical industry since affected people have limited financial resources. Although financial return on investment is insufficient for drug discovery process and development, there is a constant desperate need for new chemical entities presenting new mechanisms of action. Higher plants, marine organisms, and microorganisms provide immense opportunities for the discovery of new drugs and drug leads. The screening of these natural sources thus remains one of the most attractive routes to discovering and developing new drugs. This article reviews the importance of natural products as a source of antiparasitic drugs and discusses some of the research challenges.

Synthesis and Evaluation of Antiplasmodial Activities of Fluorinated 6-Amino- 2-Aryl-3H-Indolone-N-Oxides

A series of novel 6-amino-2-aryl-3H-indolone-N-oxides were synthesized at yields of up to 65% and characterized; one was further characterized using X-ray crystallographic analysis. Synthesized compounds were evaluated for their in vitro activity against a chloroquine-resistant (FcB1) strain of Plasmodium falciparum, as well as for the 50% cytotoxic concentration (CC50) on Vero cell lines. The most promising activities were observed for the fluorinated compounds, the most active in vitro being 6-(2-morpholinoacetamido)-2-(4-(trifluoromethoxy) phenyl)-3H-indolone-N-oxide (IC50: 15.5 nM). In addition, these compounds showed weak cytotoxicity leading to selectivity index values of >170, thus warranting further in vitro and in vivo studies.