Target sequencing approach intended to discover new mutations in non-syndromic intellectual disability.

The technological improvements over the last years made considerable progresses in the knowledge of the etiology of intellectual Disability (ID). However, at present very little is known about the genetic heterogeneity underlying the non-syndromic form of ID (NS-ID). To investigate the genetic basis of NS-ID we analyzed 43 trios and 22 isolated NS-ID patients using a targeted sequencing (TS) approach. 71 NS-ID genes have been selected and sequenced in all subjects. We found putative pathogenic mutations in 7 out of 65 patients. The pathogenic role of mutations was evaluated through sequence comparison and structural analysis was performed to predict the effect of alterations in a 3D computational model through molecular dynamics simulations. Additionally, a deep patient clinical re-evaluation has been performed after the molecular results. This approach allowed us to find novel pathogenic mutations with a detection rate close to 11% in our cohort of patients. This result supports the hypothesis that many NS-ID related genes still remain to be discovered and that NS-ID is a more complex phenotype compared to syndromic form, likely caused by a complex and broad interaction between genes alterations and environment factors.

Smoothened (SMO) receptor mutations dictate resistance to vismodegib in basal cell carcinoma.

Basal cell carcinomas (BCCs) and a subset of medulloblastomas are characterized by loss-of-function mutations in the tumor suppressor gene, PTCH1. PTCH1 normally functions by repressing the activity of the Smoothened (SMO) receptor. Inactivating PTCH1 mutations result in constitutive Hedgehog pathway activity through uncontrolled SMO signaling. Targeting this pathway with vismodegib, a novel SMO inhibitor, results in impressive tumor regression in patients harboring genetic defects in this pathway. However, a secondary mutation in SMO has been reported in medulloblastoma patients following relapse on vismodegib to date. This mutation preserves pathway activity, but appears to confer resistance by interfering with drug binding. Here we report for the first time on the molecular mechanisms of resistance to vismodegib in two BCC cases. The first case, showing progression after 2 months of continuous vismodegib (primary resistance), exhibited the new SMO G497W mutation. The second case, showing a complete clinical response after 5 months of treatment and a subsequent progression after 11 months on vismodegib (secondary resistance), exhibited a PTCH1 nonsense mutation in both the pre- and the post-treatment specimens, and the SMO D473Y mutation in the post-treatment specimens only. In silico analysis demonstrated that SMO(G497W) undergoes a conformational rearrangement resulting in a partial obstruction of the protein drug entry site, whereas the SMO D473Y mutation induces a direct effect on the binding site geometry leading to a total disruption of a stabilizing hydrogen bond network. Thus, the G497W and D473Y SMO mutations may represent two different mechanisms leading to primary and secondary resistance to vismodegib, respectively.

Synthesis, pharmacological evaluation, and σ1 receptor interaction analysis of hydroxyethyl substituted piperazines.

Starting from (S)- or (R)-aspartate, three synthetic strategies were explored to prepare hydroxyethyl substituted piperazines with different substituents at the N-atoms. σ receptor affinity was recorded using receptor material from both animal and human origin. σ1 affinities determined with guinea pig brain and human RPMI 8226 tumor cell lines differed slightly but showed the same tendency. (S)-2-[4-(Cyclohexylmethyl)-1-(naphthalene-2-ylmethyl)piperazin-2-yl]ethanol (7c) revealed the highest affinity at human σ1 receptors (Ki = 6.8 nM). The potent σ1 receptor ligand 7c was able to inhibit selectively the growth of three human tumor cell lines with IC50 values in the low micromolar range. The reduced growth of the RPMI-8226 cell line was caused by apoptosis. The interaction of 7c with the σ1 receptor was analyzed in detail using the 3D homology model of the σ1 receptor. The calculated free binding energies of all hydroxyethylpiperazines nicely correlate with their recorded affinities toward the human σ1 receptor.

Chemical, pharmacological, and in vitro metabolic stability studies on enantiomerically pure RC-33 compounds: promising neuroprotective agents acting as σ1 receptor agonists.

Our recent research efforts identified racemic RC-33 as a potent and metabolically stable σ1 receptor agonist. Herein we describe the isolation of pure RC-33 enantiomers by chiral chromatography, assignment of their absolute configuration, and in vitro biological studies in order to address the role of chirality in the biological activity of these compounds and their metabolic processing. The binding of enantiopure RC-33 to the σ1 receptor was also investigated in silico by molecular dynamics simulations. Both RC-33 enantiomers showed similar affinities for the σ1 receptor and appeared to be almost equally effective as σ1 receptor agonists. However, the R-configured enantiomer showed higher in vitro hepatic metabolic stability in the presence of NADPH than the S enantiomer. Overall, the results presented herein led us to select (R)-RC-33 as the optimal candidate for further in vivo studies in an animal model of amyotrophic lateral sclerosis.

Are two better than one? A novel double-mutant KIT in GIST that responds to Imatinib.

Gastrointestinal stromal tumors carry in about 85% of the cases activating mutations in KIT gene. Generally only one KIT mutation is found in primary tumors and the majority of mutations affecting KIT exon 11 is sensitive to Imatinib. We report upon a GIST case harboring a double-mutant KIT gene at exon 11, which expresses a receptor bearing the known activating W557G mutation and a newly discovered missense Y578C alteration. The relative affinities for ATP and Imatinib of each single (W557G, Y578C) and double (W557G/Y578C) mutant KITs were predicted by in silico studies (computer-based molecular simulations), and compared with those obtained for known Imatinib sensitive and resistant KIT mutants. In parallel, biochemical analysis of the single and double KIT mutants expressed in mammalian cells was performed. Both the in-silico/in-vitro investigations showed constitutive activation and sensitivity to Imatinib of the yet mentioned Y578C mutation as well as of the double mutant, providing evidence that the concomitant presence of the W557G and Y578C mutations does not affect Imatinib response compare to the single mutations, in line with what observed in Imatinib treated patient.

Another brick in the wall. Validation of the σ1 receptor 3D model by computer-assisted design, synthesis, and activity of new σ1 ligands.

Originally considered an enigmatic polypeptide, the σ(1) receptor has recently been identified as a unique ligand-regulated protein. Many studies have shown the potential of σ(1) receptor ligands for the treatment of various diseases of the central nervous system (CNS); nevertheless, almost no information about the 3D structure of the receptor and/or the possible modes of interaction of the σ(1) protein with its ligands have been unveiled so far. With the present work we validated our σ(1) 3D homology model and assessed its reliability as a platform for σ(1) ligand structure-based drug design. To this purpose, the 3D σ(1) model was exploited in the design of 33 new σ(1) ligands and in their ranking for receptor affinity by extensive molecular dynamics simulation-based free energy calculations. Also, the main interactions involved in receptor/ligand binding were analyzed by applying a per residue free energy deconvolution and in silico alanine scanning mutagenesis calculations. Subsequently, all compounds were synthesized in our laboratory and tested for σ(1) binding activity in vitro. The agreement between in silico and in vitro results confirms the reliability of the proposed σ(1) 3D model in the a priori prediction of the affinity of new σ(1) ligands. Moreover, it also supports and corroborates the currently available biochemical data concerning the σ(1) protein residues considered essential for σ(1) ligand binding and activity.

Pd-catalyzed direct C-H bond functionalization of spirocyclic σ1 ligands: generation of a pharmacophore model and analysis of the reverse binding mode by docking into a 3D homology model of the σ1 receptor.

To explore the hydrophobic binding region of the σ(1) receptor protein, regioisomeric spirocyclic thiophenes 9-11 were developed as versatile building blocks. Regioselective α- and ß-arylation using the catalyst systems PdCl(2)/bipy/Ag(2)CO(3) and PdCl(2)/P[OCH(CF(3))(2)](3)/Ag(2)CO(3) allowed the introduction of various aryl moieties at different positions in the last step of the synthesis. The increasing σ(1) affinity in the order 4 < 5/6 < 7/8 indicates that the positions of the additional aryl moiety and the S atom in the spirocyclic thiophene systems control the σ(1) affinity. The main features of the pharmacophore model developed for this class of σ(1) ligands are a positive ionizable group, a H-bond acceptor group, two hydrophobic moieties, and one hydrophobic aromatic group. Docking of the ligands into a σ(1) 3D homology model via molecular mechanics/Poisson-Boltzmann surface area calculations led to a very good correlation between the experimentally determined and estimated free energy of receptor binding. These calculations support the hypothesis of a reverse binding mode of ligands bearing the aryl moiety at the "top" (compounds 2, 3, 7, and 8) and "left" (compounds 4, 5, and 6) positions, respectively.

2-difluoromethylene-4-methylenepentanoic acid, a paradoxical probe able to mimic the signaling role of 2-oxoglutaric acid in cyanobacteria.

2-Difluoromethylene-4-methylenepentanoic acid (DFMPA), a seemingly deviated analog of 2-oxoglutaric acid (2-OG), could surprisingly mimic its signaling function in cyanobacteria. Computer modeling revealed the favorable binding of DFMPA toward the 2-OG receptor, NtcA, via mutual conformational changes, suggesting that structural alteration of 2-OG is tolerated for it to exercise its signaling role. This extremely useful finding could be exploited for the design of affinity probes with which to study new 2-OG receptors in related signaling pathways.