4 research outputs found
Fluorescent Derivatives of AC-42 To Probe Bitopic Orthosteric/Allosteric Binding Mechanisms on Muscarinic M1 Receptors
Two fluorescent derivatives of the M1 muscarinic selective
agonist
AC-42 were synthesized by coupling the lissamine rhodamine B fluorophore
(in ortho and para positions) to AC42-NH<sub>2</sub>. This precursor,
prepared according to an original seven-step procedure, was included
in the study together with the LRB fluorophore (alone or linked to
an alkyl chain). All these compounds are antagonists, but examination
of their ability to inhibit or modulate orthosteric [<sup>3</sup>H]NMS
binding revealed that para-LRB-AC42 shared several properties with
AC-42. Carefully designed experiments allowed para-LRB-AC42 to be
used as a FRET tracer on EGFP-fused M1 receptors. Under equilibrium
binding conditions, orthosteric ligands, AC-42, and the allosteric
modulator gallamine behaved as competitors of para-LRB-AC42 binding
whereas other allosteric compounds such as WIN 51,708 and <i>N</i>-desmethylclozapine were noncompetitive inhibitors. Finally,
molecular modeling studies focused on putative orthosteric/allosteric
bitopic poses for AC-42 and para-LRB-AC42 in a 3D model of the human
M1 receptor
Quantitative Structure–Property Relationship Modeling: A Valuable Support in High-Throughput Screening Quality Control
Evaluation
of important pharmacokinetic properties such as hydrophobicity
by high-throughput screening (HTS) methods is a major issue in drug
discovery. In this paper, we present measurements of the chromatographic
hydrophobicity index (CHI) on a subset of the French chemical library
Chimiothèque Nationale (CN). The data were used in quantitative
structure–property relationship (QSPR) modeling in order to
annotate the CN. An algorithm is proposed to detect problematic molecules
with large prediction errors, called outliers. In order to find an
explanation for these large discrepancies between predicted and experimental
values, these compounds were reanalyzed experimentally. As the first
selected outliers indeed had experimental problems, including hydrolysis
or sheer absence of expected structure, we herewith propose the use
of QSPR as a support tool for quality control of screening data and
encourage cooperation between experimental and theoretical teams to
improve results. The corrected data were used to produce a model,
which is freely available on our web server at http://infochim.u-strasbg.fr/webserv/VSEngine.html
New Fluorescein Precursors for Live Bacteria Detection
Swiftness,
reliability, and sensitivity of live bacteria detection
in drinking water are key issues for human safety. The most widespread
used indicator of live bacteria is a caged form of carboxyfluorescein
in which 3′ and 6′ hydroxyl groups are masked as acetate
esters (CFDA). This derivatization altogether abolishes fluorescein
fluorescence and renders the molecule prone to passive diffusion through
bacterial membranes. Once in the cytoplasm, acetate groups from CFDA
are removed by bacterial hydrolases and fluorescence develops, rendering
live but not dead cells detectable. Yet the reagent, carboxyfluorescein
diacetate, still possesses a free carboxyl group whose ionization
constant is such that the majority of the probe is charged at physiological
pH. This unfavors probe permeation through membranes. Here, we prepare
several chemical modifications of the carboxyl moiety of CFDA, in
order to neutralize its charge and improve its passive diffusion through
membranes. We show that the ethylamido derivative of the 5-carboxyl
group from 5-carboxy-fluorescein diacetate or from Oregon green diacetate
or from Oregon green diacetoxymethylester are stable molecules in
biological media, penetrate into bacterial cells and are metabolized
into fluorescent species. Only live bacteria are revealed since bleached
samples are not labeled. Other derivatives with modification of the
5-carboxyl group with an ester group or with a thiourea-based moiety
were almost inefficient probes. The most interesting probe, triembarine
(5-ethylaminocarboxy-oregon green, 3′,6′diacetoxymethyl
ester) leads to 6–10 times more sensitive detection of bacteria
as compared to CFDA. Addition of contrast agents (trypan blue or brilliant
blue R) improve the signal-to-noise ratio by quenching extracellular
fluorescence while bromophenol blue quenches both intracellular and
extracellular fluorescence, allowing standardization of detections
Prodrugs of a CXC Chemokine-12 (CXCL12) Neutraligand Prevent Inflammatory Reactions in an Asthma Model in Vivo
Chalcone 4 (compound <b>1</b>) is a small molecule
that neutralizes
the CXC chemokine CXCL12 and prevents it from acting on the CXCR4
and CXCR7 receptors. To overcome its poor solubility in aqueous buffers,
we designed highly soluble analogues of compound <b>1</b>, phosphate, l-seryl, and sulfate, all inactive by themselves on CXCL12 but
when cleaved in vivo into <b>1</b>, highly active locally at
a low dose in a mouse airway hypereosinophilia model