Synthesis and Biological Evaluation of New Creatine Fatty Esters Revealed Dodecyl Creatine Ester as a Promising Drug Candidate for the Treatment of the Creatine Transporter Deficiency

The creatine transporter deficiency is a neurological disease caused by impairment of the creatine transporter SLC6A8, resulting in mental retardation associated with a complete absence of creatine within the brain and cellular energy perturbation of neuronal cells. One of the therapeutic hypotheses was to administer lipophilic creatine derivatives which are (1) thought to have better permeability through the cell membrane and (2) would not rely on the activity of SLC6A8 to penetrate the brain. Here, we synthesized creatine fatty esters through original organic chemistry process. A screening on an in vitro rat primary cell-based blood–brain barrier model and on a rat primary neuronal cells model demonstrated interesting properties of these prodrugs to incorporate into endothelial, astroglial, and neuronal cells according to a structure–activity relationship. Dodecyl creatine ester showed then a 20-fold increase in creatine content in pathological human fibroblasts compared with the endogenous creatine content, stating that it could be a promising drug candidate

Expression levels of wild type, mutated UCP2 and UCP1 in mitochondrial extracts isolated from insulinoma INS-1E cells after adenoviral infection.

<p>Gels were loaded with 10 µg of mitochondrial proteins and analyzed by Western blot. A) INS-1E cells were infected with control adenovirus (Adcontrol, containing the GFP gene and the inverted sequence of human UCP2 cDNA at 5.2×10⁶ plaque-forming units (pfu)/ml) or with wild type (AdhUCP2 at 5.2×10⁶ pfu/ml) or mutated (AdhU2Patient1 at 1.3×10⁶ pfu/ml and AdhU2Patient2 at 2.6×10⁶ pfu/ml) UCP2 adenovirus. Mitochondria-rich fraction from not infected INS-1E cells was also used as control of endogenous UCP2 expression. 1 µg of mitochondrial proteins from yeasts overexpressing human UCP2 (pYeDP-hUCP2) were used as positive control. UCP2 protein was revealed using anti-hUCP2 antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003850#pone.0003850-Pecqueur1" target="_blank">[31]</a> and anti-UCP2 (C-20) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). As a control of similar amount of proteins loaded, the membrane was reprobed with anti-VDAC/Porin antibody (Sigma-Aldrich, St Louis, MO, USA). B) INS-1E cells were infected with control (Adcontrol, containing the GFP gene and the inverted sequence of human UCP2 cDNA) or with rat UCP1 adenovirus (AdrUCP1) both at a concentration of 5.2×10⁶ pfu/ml. Mitochondria-rich fraction from not infected INS-1E cells was also used as control of endogenous expression. 40 ng of purified rat UCP1 were used as positive control. UCP1 protein was revealed using a polyclonal anti-rat UCP1 antibody prepared in the laboratory. As a control of similar amount of proteins loaded, the membrane was reprobed with anti-VDAC/Porin antibody (Sigma-Aldrich, St Louis, MO, USA).</p

Comparison of the proton leak kinetics of yeast spheroplasts.

<p>Spheroplasts were isolated from control (pYeDP plasmid, black circles), wild type UCP2 (pYeDP-hUCP2, black triangles) and mutated (as found in patient 1 and 2) UCP2 (pYeDP-hU2Patient1, open diamonds and pYeDP-hU2Patient2, grey squares) yeasts as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003850#s2" target="_blank">Materials and Methods</a>” section. Mitochondrial membrane potential and respiratory rate were simultaneously recorded and varied by titration with potassium cyanide in the presence of NADH. These experiments were performed in the presence of oligomycin using the same amount of respiratory chain for the different types of spheroplasts as determined from the maximal respiratory rate in the presence of FCCP (for details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003850#s2" target="_blank">Materials and Methods</a>” section). Respiratory rate (<i>y</i> axis) is represented as the percentage from the maximal respiratory rate (Vmax) in the presence of FCCP. Membrane potential (<i>x</i> axis) is represented as the percentage from the highest membrane potential in state 4 of respiration. Results are means±S.E.M. of three independent experiments performed at least in duplicate.</p

Pedigree of Families 1 and 2 with missense heterozygous sequence variations in the UCP2 gene.

<p>A. Pedigree: Family 1 (F1): Patient 1 had neonatal hypoglycaemia, was sensitive to diazoxide and resolved spontaneously at age 1 year. Her parents did not present clinical signs of hypoglycaemia. Family 2 (F2): Patient 2 developed hypoglycaemia at 8 month of age, sensitive to diazoxide. His mother presented seizures in infancy and occasional hypoglycaemia. Individuals carrying heterozygous <i>UCP2</i> sequence variations are indicated by partly closed and partly open symbols. B. DHPLC patterns (upper part) and corresponding DNA sequences (lower part) of family 1 and family 2.</p

Amino acid changes in UCP2 of patients 1 and 2 and conservation among species.

<p>Comparison of the aminoacid sequences of the second moiety of UCP2 from <i>Homo sapiens</i> (GenBank Accession NP003346), <i>Rattus norvegicus</i> (GenBank Accession NP062227), <i>Phodopus sungorus</i>(GenBank Accession AAG33984), <i>Mus musculus</i> (GenBank Accession NP035801), <i>Canis familiaris</i> (GenBank Accession BAA90457), <i>Sus scrofa</i> (GenBank Accession NP999454), <i>Cyprinus carpio</i> (GenBank Accession CAB46248), <i>Danio rerio</i> (GenBank Accession NP571251). The multiple sequence alignment was carried out with Clustal X (v. 1.8) analysis software. The UCP2 consensus sequence was calculated from the site <a href="http://coot.embl.de/cgi/consensus" target="_blank">http://coot.embl.de/cgi/consensus</a>. The ribbon cartoon symbolizes the predicted α-helix domains according to the 3D structure of the ADP/ATP carrier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003850#pone.0003850-PebayPeyroula1" target="_blank">[51]</a>. The putative transmembrane motifs are marked in grey background and the residues mutated in the two patients in dark grey background. Bold letters in the upper line represent the aminoacids found in Patient 1 and Patient 2.</p

PCR an DHPLC conditions

<p>PCR an DHPLC conditions</p

Effect of UCP2 mutations on insulin secretion of INS-1E cells in response to glucose.

<p>Insulin release by INS-1E cells expressing adenoviral wild type (AdhUCP2), mutated (AdhU2Patient1 and AdhU2Patient2) UCP2 or rat UCP1 (AdrUCP1) compared to control (Adcontrol, containing the GFP gene and the inverted sequence of human UCP2 cDNA). Values represent insulin secreted into the medium as percentage from the insulin secreted by cells infected with control adenovirus (Adcontrol) in the presence of 15 mM glucose (for details see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003850#s2" target="_blank">Materials and Methods</a>” section). 32 µM retinoic acid (RA) was added to the cultures of AdUCP1 infected cells to activate UCP1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003850#pone.0003850-Rial1" target="_blank">[36]</a>. Data are means±S.E.M. of at least 6 independent experiments performed in duplicate. The level of statistical significance of the differences between cells infected with control adenovirus and with UCP2 or UCP1 adenovirus is indicated: ** <i>p</i><0.001 (<i>t</i> test).</p

1A: Family tree showing the 3 affected children.

<p>1B: Crystal structure of human muscle aldolase complexed with fructose 1,6-bisphosphate (isoenzyme A, PDB code 4ALD) superimposed with the tetrameric crystal structure of human brain aldolase (isoenzyme C, PDB code 1XFB), which is similar to the muscle isoenzyme. Chains A, B, C and D of isoenzyme C are shown in orange, light blue, light green and pink, respectively. Monomeric isoenzyme A is shown in grey and is superimposed on chain D of the tetrameric isoenzyme C. Fructose 1,6-bisphosphate co-crystallized with isoenzyme A is shown in yellow. The mutated residue described in this report (red arrow) and the mutated amino acids previously described are highlighted in the magnified structure. The structural and functional consequences of the mutations are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004711#pgen-1004711-t001" target="_blank">Table 1</a>. 1C: aldolase A, glucose-6-phosphate dehydrogenase (G6PD) and hexokinase activities in the erythrocytes of the parents, the healthy sibling and the 3 affected patients (*: patients 2, 3, 4). 1D: in vitro muscle study of anaerobic glycogenolysis and glycolysis (only patient 3); results of lactate production (µmol/g muscle in 30 minutes) after incubation with various substrates.</p

Reported cases of Aldolase A deficiency with the described mutations.

<p>*: normal range. In the first case reported by Beutler et al in 1973 with no described mutation, the red cell aldolase activity was 16% of normal mean.</p><p>Reported cases of Aldolase A deficiency with the described mutations.</p

<i>ALDOA</i> expression and activity.

<p>3A:<i>ALDOA</i> mRNA expression in control myoblasts (C, white bars) and the patient myoblasts (P, grey bars) under basal conditions, with TNFα+Ilβ treatment (left) or at a high temperature (right, 40°C); Aldolase A protein levels (lower panel) under basal conditions, with TNFα+Ilβ treatment or at a high temperature. 3B: Aldolase A activity in control and the patients' myoblasts under the same conditions: basal conditions, TNFα+Ilβ treatment and at different temperatures. The results are shown as the mean value ±SD from 3 independent experiments. 3C: Aldolase A activity in control and patients erythrocytes under basal conditions and at different temperatures. The results are shown as the mean value of two independent experiments. 3D: Aldolase A activity (upper) and protein level (below) in the patient myoblasts under basal condition and after arginine (Arg) treatment.*: p<0,05).</p