The design and synthesis of inhibitors of Mycobacterium tuberculosis thymidylate kinase (MtTMPK)

Thymidylate kinase (TMPK) phosphorylates thymidine 5’-monophosphate (dTMP) and has been proposed as an attractive target for Mycobacterium tuberculosis (Mt).1 By mimicking the structure of the substrate (dTMP), we have previously discovered different series of nucleoside analogues with MtTMPK inhibitory activities in a micromole range.2 Based on recently reported potent piperidin-3-yl-thymine inhibitors of Gram-positive bacterial TMPK,3 we report a series of isomeric N-benzyl-substituted piperidin-4-yl-thymine analogues, some of which demonstrate potent Mt TMPK inhibitory activity. Towards this end a convenient and high-yield synthesis was developed to access 1-substitued thymine derivatives

Elaboration of a proprietary thymidylate kinase inhibitor motif towards anti-tuberculosis agents

International audienceWe report the design and synthesis of a series of non-nucleoside MtbTMPK inhibitors (1-14) based on the gram-positive bacterial TMPK inhibitor hit compound 1. A practical synthesis was developed to access these analogues. Several compounds show promising MtbTMPK inhibitory potency and allow the establishment of a structure-activity relationship, which is helpful for further optimization

1-(1-Arylethylpiperidin-4-yl)thymine analogs as antimycobacterial TMPK inhibitors

A series of Mycobacterium tuberculosis TMPK (MtbTMPK) inhibitors based on a reported compound 3 were synthesized and evaluated for their capacity to inhibit MtbTMPK catalytic activity and the growth of a virulent M. tuberculosis strain (H37Rv). Modifications of the scaffold of 3 failed to afford substantial improvements in MtbTMPK inhibitory activity and antimycobacterial activity. Optimization of the substitution pattern of the D ring of 3 resulted in compound 21j with improved MtbTMPK inhibitory potency (three-fold) and H37Rv growth inhibitory activity (two-fold). Moving the 3-chloro substituent of 21j to the para-position afforded isomer 21h, which, despite a 10-fold increase in IC50-value, displayed promising whole cell activity (minimum inhibitory concentration (MIC) = 12.5 μM)

Synergy of the antibiotic colistin with echinocandin antifungals in Candida species.

International audienceOBJECTIVES: Candida albicans is the most prevalent fungal pathogen of humans, causing a wide range of infections from harmless superficial to severe systemic infections. Improvement of the antifungal arsenal is needed since existing antifungals can be associated with limited efficacy, toxicity and antifungal resistance. Here we aimed to identify compounds that act synergistically with echinocandin antifungals and that could contribute to a faster reduction of the fungal burden. METHODS: A total of 38 758 compounds were tested for their ability to act synergistically with aminocandin, a β-1,3-glucan synthase inhibitor of the echinocandin family of antifungals. The synergy between echinocandins and an identified hit was studied with chemogenomic screens and testing of individual Saccharomyces cerevisiae and C. albicans mutant strains. RESULTS: We found that colistin, an antibiotic that targets membranes in Gram-negative bacteria, is synergistic with drugs of the echinocandin family against all Candida species tested. The combination of colistin and aminocandin led to faster and increased permeabilization of C. albicans cells than either colistin or aminocandin alone. Echinocandin susceptibility was a prerequisite to be able to observe the synergy. A large-scale screen for genes involved in natural resistance of yeast cells to low doses of the drugs, alone or in combination, identified efficient sphingolipid and chitin biosynthesis as necessary to protect S. cerevisiae and C. albicans cells against the antifungal combination. CONCLUSIONS: These results suggest that echinocandin-mediated weakening of the cell wall facilitates colistin targeting of fungal membranes, which in turn reinforces the antifungal activity of echinocandins

Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation†

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(–) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified

Eight previously unidentified mutations found in the OA1 ocular albinism gene

BACKGROUND: Ocular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. METHODS: The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. RESULTS: We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. CONCLUSION: The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand

Leber Congenital Amaurosis: Comprehensive Survey of the Genetic Heterogeneity, Refinement of the Clinical Definition, and Genotype-Phenotype Correlations as a Strategy for Molecular Diagnosis

Communicated by Jean-Claude Kaplan Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rodcone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth

Comment conceptualiser la hauteur du soleil en tant qu'angle au cycle 3 ?

How to conceptualise the height of the sun by an angle with 10-11 year old pupils Hélène Merle Valérie Munier The height of the sun is often likened to a length although the astronomical term refers to the angle between the direction of the sun and the horizontal plane. With 10-11 year-old pupils this concept proves to have an additional difficulty linked to the concept of angle. We have presented the problem of identifying the height of the sun to four classes and compared two methods currently employed : the method of the shadow made on the floor by a vertical object and the method of aiming directly at the sun. The analysis and subsequent evaluation of these sequences show that the majority of students are capable of identifying the height of the sun as an angle even when this identification is left to their own resources. Also the method of aiming directly at the sun, introduces the pupils to the construction of an instrument which can measure the height of the sun and to its use. It is this method which turns out to be more pertinent to achieve the objective.La hauteur du Soleil est souvent assimilée à une longueur alors qu’elle désigne, en astronomie, l’angle que fait la direction de visée du Soleil avec le plan horizontal. Avec des élèves de 10-11 ans, cette conception se double d’une difficulté supplémentaire liée au concept d’angle. Nous avons confronté quatre classes de cours moyen au problème du repérage de la hauteur du Soleil et comparé deux méthodes couramment employées dans les classes : la méthode de l’ombre portée sur le sol d’un objet vertical et la méthode de visée directe du Soleil. L’analyse des séquences et des évaluations a posteriori montre d’une part que la majorité des élèves est capable d’identifier la hauteur du Soleil à un angle, alors même que cette identification était laissée à leur charge. D’autre part la méthode de visée, qui confronte les élèves à la conception d’un instrument de mesure de la hauteur du Soleil puis à son utilisation, s’avère plus pertinente pour atteindre l’objectif visé.Como conceptualizar la altura del sol en tanto que ángulo con alumnos de 10-11 años ? Hélène Merle Valérie Munier La altura del sol es a menudo asimilada a una longitud que en tal caso representa, en astronomía, el ángulo que hace la dirección que apunta el sol con el plano horizontal. Con los alumnos de 10-11 años esta concepción tiene una dificultad suplementaria ligada al concepto de ángulo. Hemos confrontado cuatro clases del curso medio al problema de la altura del Sol y comparado dos métodos corrientement empleados en las clases : el método de la sombra llevada sobre el piso de un objeto vertical y el método que apunta directamente al Sol. El análisis de las secuencias y evaluaciones a posteriori muestra de una parte que la mayoría de los alumnos es capaz de identificar la altura del Sol en un ángulo, mismo que esa identificación fué dejada a su cargo. Por otra parte el método que confronta los alumnos a la concepción de un instrumento de medida de la altura del Sol, luego de su utilización, es más pertinente para alcanzar el objetivo enfocado.Merle Hélène, Munier Valérie. Comment conceptualiser la hauteur du soleil en tant qu'angle au cycle 3 ?. In: Aster, recherches en didactique des sciences expérimentales, n°36, 2003. L'enseignement de l'astronomie. pp. 39-68