AnnualReport2020

COORDINATION COMPOUNDS AS ANTIOXIDANTS:
ACTIVITY EVALUATION BY COMBINING FIRST-
PRINCIPLE CALCULATIONS AND SOLID-STATE NMR
THE ROLE OF PARASITE DERIVED P-GLYCOPROTEINS
AS MEDIATORS FOR ANTHELMINTIC RESISTANCE
Dr Arlette Richaud-Torres
LE STUDIUM / Marie Skłodowska-Curie
Research Fellow
Smart Loire Valley General Programme
From: Autonomous Metropolitan
University Unidad Iztapalapa - MX
In residence at: Extreme Conditions
and Materials: High Temperature and
Irradiation (CEMHTI) - Orléans
Nationality: Mexican
Dates: June 2019 to May, 2020
I am Chemist by UNAM (Mexico) and obtained a
PhD in Sciences (Chemistry) by UAM (Mexico).
I have expertise on synthetic strategies of
coordination and organic compounds for
biological aplications as well as in analytical
and spectroscopic methodologies for structural
characterisation of those compounds and
theoretical reactivity studies of different chemical
systems, specially in heterocyclic compounds
in the design of biological active compounds.
I have been referee of international journals,
professor chemistry and environemental legacy
at UAM (Mexico). I worked as invited professor
at Donostia International Physics Center (Spain),
Euskal Herriko Unibertsitatea (Spain), Valladolid
University (Spain), Marie Skłodowska-Curie
Research Fellow by Le Studium. I am CONACyT-
Mexico National Researcher (SNI level I) and
funder and organiser of the NMR symposia in
Mexico and actually, researcher at CNRS- CEMHTI.
To develop extra thermodynamic relationships between chemical reactivity
parameters and structural data obtained by solid state NMR of coordination
compounds to follow their antioxidant ability.
Twenty coordination compounds were obtained by the use of different
inorganic salts of divalent transition metal cations with the 4(5)-methyl
imidazole ligand. The compounds were characterized by analytical and
spectroscopical methods. The experimental 1 H, 13 C and 15 N Solid State
NMR spectra for each compound were also determined. Some calculus at
first principles were based in the structural data obtained by single crystal
X-ray diffraction and some others by the structural interpretation of the
spectroscopical NMR studies. The structural information obtained was
used to calculate the chemical reactivity parameters based in the Hard and
Soft Acids and Basis Principle (HSAB) supported by the Density Functional
Theory (DFT) theory. The isotropic chemical displacement observed in the
experimental 1 H, 13 C and 15 N solid state NMR spectra were in agreement with
the electronic density characteristics revealed by the reactivity parameters
obtained for each site in the testing molecules.
The results of the first principle calculations of NMR parameters were also in
agreement with their respective experimental NMR spectra. The analysis of
the theoretical and experimental results allow us to identify the influence of
the substituent in the ligands as well as in the formation of the coordination
compounds and their influence in the antioxidant behavior of the nickel,
cobalt and copper compounds. The influence of the counteranion was
evidenced and the correlation of the antioxidant ability of each compound
was correlated with the 1 H and 13 C isotropic values.
We have obtained different correlations between the HSAB-DFT reactivity
parameters of the designed and synthetized coordination compounds
related to its antioxidant behavior and its solid state NMR parameters.
The relationships between the electrophilicity and the proton and carbon
ss-NMR isotropic values presented better correlation in comparison to the
data for 15 N.
Prof. Georg von Samson-
Himmelstjerna
LE STUDIUM Guest Research Fellow
From: Free University of Berlin - DE
In residence at: Infectiology and Public
Health (ISP) - Tours
Nationality: German
Dates: October 2019 to March 2020
Following his degree in veterinary medicine Georg
von Samson-H. obtained a Dr med. vet. degree in
veterinary parasitology and then spent four years
as laboratory head in the pharmaceutical industry
before returning to academia.
He successfully pursued his research carreed in
veterinary parasitology by receiving his habilitation
in 2003 and his first full professor position at the
Institut of Parasitology, University of Veterinary
Medicine in Hannover, Germany in 2005.
Since 2009 he is University Professor at the
Institute for Parasitology and Tropical Veterinary
Medicine, Freie Universität Berlin, Germany.
His main research topics include mechanisms
of anthelmintics and the occurrence as well
as molecular mechanisms of anthelmintic
resistance.
He has published over 200 scientific papers in
journals with peer review (current web of science
H-index: 37, sum of citations: 5242) and attracted
over 70 extramurally funded research projects.
The main objectives of the Host laboratory at INRAE Centre Val de Loire
(Multiresistance and pathogeny factors from parasitic nematodes headed
by Cedric Neveu) and the Institute for Parasitology and Tropical Veterinary
Medicine (IPTVM) at Freie Universität Berlin (headed by Prof. Georg von
Samson-Himmelstjerna) are innovative contributions to the sustainable
control of gastro-intestinal parasitic nematodes (GIN) in livestock industry.
Currently, the control of GIN infections remains largely based on anthelmintic
treatments, but increasing anthelmintic resistance has reduced their efficacy.
In that respect, during the past decade, both groups have investigated
anthelmintic resistance (AR) on several different aspects from field studies
to molecular characterization of parasite anthelmintic targets.
The current project focusses on the investigation of metabolic pathways of
anthelmintics, in particular the P-glycoproteins (Pgp) and their mutations in
the context of drug transport, specifically concerning macrocyclic lactones
(ML). As it is to date not feasible to perform forward or reverse genetic
studies in parasitic nematodes and thus also not possible to study the effects
of gene modifications directly in these organisms, scientists employ model
organisms such as the earth nematode Caenorhabditis elegans in which
the respective parasite derived genes are being functionally expressed.
Specifically the Pgp11 gene of the equine GIN Parascaris univalens was
cloned into wild-type as well as Pgp11 knock-out lines of C. elegans. This
was performed using the CRISPR/Cas9 technology which is well established
in the host institution. Based on previous findings in the laboratory of the Le
Studium fellow there is circumstantial evidence showing that specific single
nucleotide polymorphisms in the Parascaris Pgp11 coding sequence are
associated with reduced ML efficacy. To investigate the functional relevance
of these polymorphisms recombinant C. elegans lines expressing Parascaris
Pgp11 with and without these polymorphisms were generated. A total of five
different CRISPR/Cas9 experiments was performed employing two Parascaris
Pgp11.1 constructs without the ML-resistance associated SNPs and two
with these. Furthermore, one transformation with the plasmid encoding for
mCherry was conducted. All experiments resulted in worms exhibiting the
roller phenotype and showing the expected antibiotic resistance. Offspring
of worms transformed using the mCherry-containing construct driving the
expected intestinal expression was confirmed using fluorescence microscopy
(Fig. 1). For the confirmation of successful integration in the correct genetic
locus a range of PCR primer pairs was designed and tested in several of
the obtained C. elegans lines. Finally, one of the primer pairs resulted in
the generation of fragments which were confirmed by Sanger sequencing to
represent the inserted mCherry encoding construct.
Dr Pierre Florian
Host Scientist
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He is an expert and international reference on Solid-
State Nuclear Magnetic Resonance, specifically on
about spectroscopy of crystalline and amorphous
phases, structure of oxide glasses, structure and
dynamics of very high–temperature molten oxides. He
has performed in joint with Dr Massiot the network
Research infrastructure Magnetic Nuclear Resonance,
Very High Fields in France. Actually, he has more than
104 peer-reviewed publications, with an average
citation per article of 28.5 and his h-index is 31. He has
co-organized and participated as lecturer in more than
20 international conferences where the high quality of
his research has been shown. Honored in 2019 with
the crystal prize by CNRS due to his relevant role in
the IR-NMR infrastructure research group. He is part
of the organizing committee of the Rocky Mountain
Conference on Magnetic Resonance (July,2020
Colorado USA) and the NMR Symposia in Mexico.
Dr Cédric Neveu
Host Scientist
Following a PhD thesis and a post doc on the
pathogenic bacteria Helicobacter pylori, Cédric
Neveu focused on plant resistance and the
mechanisms by which the parasitic nematode
Meloidogyne incognita overcomes these defences.
He was recruited to the French National Institute
for Agricultural Research (Inra) in 2005. Currently,
he is in charge of a research team working on
gastro-intestinal parasitic nematodes of small
ruminants. The main focus of his research work
concerns identification of molecular mechanisms
involved in anthelmintic resistance and the
development of alternative control strategies.
Figure 1. Two tm0333 worms showing intestinal red fluorescence based
on expression of mCherry and exhibiting the roller phenotype.
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