Nanodiscs for INPHARMA NMR Characterization of GPCRs: Ligand Binding to the Human A2A Adenosine Receptor.

G-protein-coupled-receptors (GPCRs) are of fundamental importance for signal transduction through cell membranes. This makes them important drug targets, but structure-based drug design (SBDD) is still hampered by the limitations for structure determination of unmodified GPCRs. We show that the interligand NOEs for pharmacophore mapping (INPHARMA) method can provide valuable information on ligand poses inside the binding site of the unmodified human A2A adenosine receptor reconstituted in nanodiscs. By comparing experimental INPHARMA spectra with back-calculated spectra based on ligand poses obtained from molecular dynamics simulations, a complex structure for A2A R with the low-affinity ligand 3-pyrrolidin-1-ylquinoxalin-2-amine was determined based on the X-ray structure of ligand ZM-241,358 in complex with a modified A2A R

De novo fragment-based design of inhibitors of DXS guided by spin-diffusion-based NMR spectroscopy

We applied for the first time an innovative ligand-based NMR methodology (STI) to a medicinal-chemistry project aimed at the development of inhibitors for the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). DXS is the first enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway, present in most bacteria (and not in humans) and responsible for the synthesis of the essential isoprenoid precursors. We designed de novo a first generation of fragments, using Deinococcus radiodurans DXS as a model enzyme, targeting the thiamine diphosphate (TDP) pocket of DXS whilst also exploring the putative substrate-binding pocket, where selectivity over other human TDP-dependent enzymes could be gained. The STI methodology – suitable for weak binders – was essential to determine the binding mode in solution of one of the fragments, circumventing the requirement for an X-ray co-crystal structure, which is known to be particularly challenging for this specific enzyme and in general for weak binders. Based on this finding, we carried out fragment growing and optimisation, which led to a three-fold more potent fragment, about as potent as the well-established thiamine analogue deazathiamine. The STI methodology proved therefore its strong potential as a tool to support medicinal-chemistry projects in their early stages, especially when dealing with weak binders

Mutation Analysis of NR5A1 Encoding Steroidogenic Factor 1 in 77 Patients with 46, XY Disorders of Sex Development (DSD) Including Hypospadias

BACKGROUND: Mutations of the NR5A1 gene encoding steroidogenic factor-1 have been reported in association with a wide spectrum of 46,XY DSD (Disorder of Sex Development) phenotypes including severe forms of hypospadias. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the frequency of NR5A1 gene mutations in a large series of patients presenting with 46,XY DSD and hypospadias. Based on their clinical presentation 77 patients were classified either as complete or partial gonadal dysgenesis (uterus seen at genitography and/or surgery, n = 11), ambiguous external genitalia without uterus (n = 33) or hypospadias (n = 33). We identified heterozygous NR5A1 mutations in 4 cases of ambiguous external genitalia without uterus (12.1%; p.Trp279Arg, pArg39Pro, c.390delG, c140_141insCACG) and a de novo missense mutation in one case with distal hypospadias (3%; p.Arg313Cys). Mutant proteins showed reduced transactivation activity and mutants p.Arg39Pro and p.Arg313Cys did not synergize with the GATA4 cofactor to stimulate reporter gene activity, although they retained their ability to physically interact with the GATA4 protein. CONCLUSIONS/SIGNIFICANCE: Mutations in NR5A1 were observed in 5/77 (6.5%) cases of 46,XY DSD including hypospadias. Excluding the cases of 46,XY gonadal dysgenesis the incidence of NR5A1 mutations was 5/66 (7.6%). An individual with isolated distal hypopadias carried a de novo heterozygous missense mutation, thus extending the range of phenotypes associated with NR5A1 mutations and suggesting that this group of patients should be screened for NR5A1 mutations

Practical consensus guidelines for the management of enuresis

Despite the high prevalence of enuresis, the professional training of doctors in the evaluation and management of this condition is often minimal and/or inconsistent. Therefore, patient care is neither optimal nor efficient, which can have a profound impact on affected children and their families. Once comprehensive history taking and evaluation has eliminated daytime symptoms or comorbidities, monosymptomatic enuresis can be managed efficaciously in the majority of patients. Non-monosymptomatic enuresis is often a more complex condition; these patients may benefit from referral to specialty care centers. We outline two alternative strategies to determine the most appropriate course of care. The first is a basic assessment covering only the essential components of diagnostic investigation which can be carried out in one office visit. The second strategy includes several additional evaluations including completion of a voiding diary, which requires extra time during the initial consultation and two office visits before treatment or specialty referral is provided. This should yield greater success than first-line treatment. Conclusion: This guideline, endorsed by major international pediatric urology and nephrology societies, aims to equip a general pediatric practice in both primary and secondary care with simple yet comprehensive guidelines and practical tools (i.e., checklists, diary templates, and quick-reference flowcharts) for complete evaluation and successful treatment of enuresis

Developmental Transcriptional Networks Are Required to Maintain Neuronal Subtype Identity in the Mature Nervous System

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply “locked-in,” but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain

Pediatric urolithiasis: the current surgical management

Children represent about 1% of all patients with urolithiasis, but 100% of these children are considered high risk for recurrent stone formation, and it is crucial for them to receive a therapy that will render them stone free. In addition, a metabolic workup is necessary to ensure a tailored metaphylaxis to prevent or delay recurrence. The appropriate therapy depends on localization, size, and composition of the calculus, as well as on the anatomy of the urinary tract. In specialized centers, the whole range of extracorporeal shock-wave lithotripsy (ESWL), ureterorenoscopy (URS), and percutaneous nephrolithotomy (PCNL) are available for children, with the same efficiency and safety as in adults