Assessment of QT liabilities in drug development

Since the publication, in 1997, of the CPMP (Committee for Proprietary Medicinal Products) Points to Consider document on "The assessment of potential for QT prolongation by non-cardiovascular medicinal products," both regulatory bodies and the pharmaceutical industry have paid increasing attention to the conduct of careful preclinical studies on the subject. Regulatory attention has focused on the drafting of Safety Pharmacology guidelines through the ICH (International Conference on Harmonization) process, which resulted in approval by the ICH and acceptance by the three main regions (USA, Europe, and Japan) of the ICH S7A guideline. The guideline does not deal only with cardiovascular studies and does not provide guidance on QT investigations. This part has been deferred to a second guideline (ICH S7B). Nevertheless, pharmaceutical companies have implemented screening strategies aimed at selecting compounds that do not present QT liabilities. These strategies can differ according to the pharmaceutical class, while experimental models differ according to the stage of development of the compound. Several in vitro models are employed in discovery (radioligand binding, high-throughput patch clamp, efflux, and fluorescence assays). These models, coupled with in silico methods, allow companies to screen a high number of compounds. Other in vitro models, applied later in the R&D process (action potential duration, APD, in Purkinje fibers or papillary muscle and the isolated heart) are useful in better describing the activity of compounds on cardiac ion channels. The most robust and accepted in vivo test is represented by telemetry studies in conscious non-rodents.     

In Vivo Identification of Photosystem II Light Harvesting Complexes Interacting with PHOTOSYSTEM II SUBUNIT S

Light is the primary energy source for photosynthetic organisms, but in excess, it can generate reactive oxygen species and lead to cell damage. Plants evolved multiple mechanisms to modulate light use efficiency depending on illumination intensity to thrive in a highly dynamic natural environment. One of the main mechanisms for protection from intense illumination is the dissipation of excess excitation energy as heat, a process called nonphotochemical quenching. In plants, nonphotochemical quenching induction depends on the generation of a pH gradient across thylakoid membranes and on the presence of a protein called PHOTOSYSTEM II SUBUNIT S (PSBS). Here, we generated Physcomitrella patens lines expressing histidine-tagged PSBS that were exploited to purify the native protein by affinity chromatography. The mild conditions used in the purification allowed copurifying PSBS with its interactors, which were identified by mass spectrometry analysis to be mainly photosystem II antenna proteins, such as LIGHT-HARVESTING COMPLEX B (LHCB). PSBS interaction with other proteins appears to be promiscuous and not exclusive, although the major proteins copurified with PSBS were components of the LHCII trimers (LHCB3 and LHCBM). These results provide evidence of a physical interaction between specific photosystem II light-harvesting complexes and PSBS in the thylakoids, suggesting that these subunits are major players in heat dissipation of excess energy.Photosynthetic organisms exploit sunlight energy to support their metabolism. However, if absorbed in excess, light can produce harmful reactive oxygen species (Li et al., 2009; Murchie and Niyogi, 2011). In a natural environment, light intensity is highly variable and can rapidly change from being limited to being in excess. To survive and thrive in such a variable habitat, plants evolved multiple strategies to modulate their light use efficiency to limit reactive oxygen species formation when exposed to excess illumination while maintaining the ability to harvest light efficiently when required (Li et al., 2009; Murchie and Niyogi, 2011; Ruban, 2015). Among these different protection processes, the fastest, called nonphotochemical quenching (NPQ), is activated in a few seconds after a change in illumination, and it leads to the thermal dissipation of excess absorbed energy. NPQ is a complex phenomenon with different components that are distinguished according to their activation/relaxation time scale (Demmig-Adams et al., 1996; Szabó et al., 2005; Niyogi and Truong, 2013). The primary and fastest NPQ component, called qE (for energy-quenching component) or feedback deexcitation, depends on the generation of a pH gradient across the thylakoid membranes (Niyogi and Truong, 2013). In land plants, qE activation requires the presence of a thylakoid protein called PHOTOSYSTEM II SUBUNIT S (PSBS; Li et al., 2000, 2004). The Arabidopsis (Arabidopsis thaliana) PSBS-depleted mutant psbs KO (Li et al., 2000) is unable to activate qE and also showed reduced fitness when exposed to natural light variations in the field, supporting a major role for this protein in responding to illumination intensity fluctuations (Li et al., 2000; Külheim et al., 2002). Mutational analyses showed that the PSBS role in qE strictly depends on the presence of two protonable Glu residues, which are most likely involved in sensing the pH decrease in the lumen (Li et al., 2004). Despite several studies, however, the precise molecular mechanism by which PSBS controls NPQ induction remains debatable, and contrasting hypotheses have been presented (for review, see Ruban et al., 2012). PSBS has been hypothesized to bind pigments and to be directly responsible for energy dissipation based on its sequence similarity with LIGHT HARVESTING COMPLEX (LHC) proteins (Li et al., 2000; Aspinall-O’Dea et al., 2002). An alternative hypothesis instead suggested that PSBS is unable to bind pigments (Funk et al., 1995; Crouchman et al., 2006; Bonente et al., 2008a) and that it plays an indirect role in NPQ by modulating the PSII antenna protein transition from light harvesting to an energy dissipative state (Betterle et al., 2009; Johnson et al., 2011). This transition has been suggested to depend on the control of the macroorganization of the PSII-LHCII supercomplexes that are present in the grana membranes (Kiss et al., 2008; Betterle et al., 2009; Kereïche et al., 2010; Johnson et al., 2011). Consistent with this hypothesis, it was recently demonstrated that PSBS is able to induce a dissipative state in isolated LHCII proteins in liposomes (Wilk et al., 2013), suggesting that its interactions with antenna proteins play a key role in its biological activity. However, the precise identity of PSBS interactors (Teardo et al., 2007; Betterle et al., 2009), the PSBS oligomerization state (Bergantino et al., 2003), and its localization within PSII supercomplexes (Nield et al., 2000; Haniewicz et al., 2013) remain unclear or at least controversial, limiting the current understanding of PSBS molecular mechanisms.The moss Physcomitrella patens has recently emerged as a valuable model organism in which to study NPQ. As in the model angiosperm Arabidopsis, PSBS accumulation modulates NPQ amplitude and protects plants from photoinhibition under strong light in P. patens (Li et al., 2000; Alboresi et al., 2010; Zia et al., 2011; Gerotto et al., 2012). PSBS-mediated NPQ in P. patens also showed zeaxanthin dependence as in other plants (Niyogi et al., 1998; Pinnola et al., 2013). The moss P. patens has another protein involved in NPQ, LHCSR, which is typically found in algae and is different from proteins found in vascular plants (Peers et al., 2009; Bailleul et al., 2010; Gerotto and Morosinotto, 2013). Even if LHCSR is present in P. patens, LHCSR- and PSBS-dependent NPQ mechanisms were shown to be independent and to have an additive effect without any significant functional synergy (Gerotto et al., 2012).Previous data also demonstrated the possibility of achieving strong overexpression of PSBS in P. patens (Gerotto et al., 2012), which, however, was never observed in Arabidopsis (Li et al., 2002). This property was exploited in this work to overexpress a His-tagged PSBS isoform, which was afterward purified in its native state from dark-adapted thylakoid membranes. Several PSII antenna proteins were copurified with PSBS and identified by mass spectrometry analyses, demonstrating that they interact physically in dark-adapted thylakoid membranes. Components of LHCII trimers (LHCB3 and LHCBM) appear to be major, but not exclusive, components of PSBS interactors.     

Differential protein–protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways

Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson's disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1‐ and LRRK2‐specific cellular processes by identifying their distinct interacting proteins. A protein microarray‐based interaction screen was performed with recombinant 3xFlag‐LRRK1 and 3xFlag‐LRRK2 and, in parallel, co‐immunoprecipitation followed by mass spectrometry was performed from SH‐SY5Y neuroblastoma cell lines stably expressing 3xFlag‐LRRK1 or 3xFlag‐LRRK2. We identified a set of LRRK1‐ and LRRK2‐specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF‐R) as a LRRK1‐specific interactor, while 14‐3‐3 proteins were LRRK2‐specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14‐3‐3 consensus binding motifs. To assess the functional relevance of these interactions, SH‐SY5Y‐LRRK1 and ‐LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14‐3‐3 binding, or with EGF, an EGF‐R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins.

     

Characterization of articular chondrocytes isolated from 211 osteoarthritic patients

We analyzed specific features of chondrocytes as cellular yield, cell doubling rates and the dependence between these parameters and patient-related data in a set of 211 osteoarthritic (OA) patients undergoing total joint replacement. For each patient the data available were joint type, age and gender. Knee samples chosen randomly among all biopsies were graded according to ICRS score. Patients’ age ranged between 30 and 90 years with a mean age of 66 ± 9.7 years. Patients were divided into age classes and statistically significant differences in proliferation rate at passage 1 were found between chondrocytes derived from young and old donors, with the last ones characterized by a lower proliferation rate. A similar trend was observed for proliferation rate at passage 2. For all the samples, cellular yields ranged between 0.1 and 5.5 million cells/g of tissue. No significant correlation was observed between the level of cartilage degeneration (ICRS score) and cellular yield and proliferation rates. However, in samples with a high degree of cartilage degeneration (ICRS score 4) the cellular yield was lower compared to the other three groups (ICRS scores 1–3). In this study we performed a systematic characterization of basic parameters of chondrocytes originating from a wide group of OA patients. Considering the use of autologous chondrocytes in chondral treatments, the characterization of cell basic features may represent an important step to determine the quality of the cell source which is a major determinant in the outcome of cell-based therapies.     

Identification of the PLK2-Dependent Phosphopeptidome by Quantitative Proteomics

Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson''s disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.     

Effects of sex chromosome dosage on corpus callosum morphology in supernumerary sex chromosome aneuploidies

Background

Supernumerary sex chromosome aneuploidies (sSCA) are characterized by the presence of one or more additional sex chromosomes in an individual’s karyotype; they affect around 1 in 400 individuals. Although there is high variability, each sSCA subtype has a characteristic set of cognitive and physical phenotypes. Here, we investigated the differences in the morphometry of the human corpus callosum (CC) between sex-matched controls 46,XY (N =99), 46,XX (N =93), and six unique sSCA karyotypes: 47,XYY (N =29), 47,XXY (N =58), 48,XXYY (N =20), 47,XXX (N =30), 48,XXXY (N =5), and 49,XXXXY (N =6).

Methods

We investigated CC morphometry using local and global area, local curvature of the CC boundary, and between-landmark distance analysis (BLDA). We hypothesized that CC morphometry would vary differentially along a proposed spectrum of Y:X chromosome ratio with supernumerary Y karyotypes having the largest CC areas and supernumerary X karyotypes having significantly smaller CC areas. To investigate this, we defined an sSCA spectrum based on a descending Y:X karyotype ratio: 47,XYY, 46,XY, 48,XXYY, 47,XXY, 48,XXXY, 49,XXXXY, 46,XX, 47,XXX. We similarly explored the effects of both X and Y chromosome numbers within sex. Results of shape-based metrics were analyzed using permutation tests consisting of 5,000 iterations.

Results

Several subregional areas, local curvature, and BLDs differed between groups.Moderate associations were found between area and curvature in relation to the spectrum and X and Y chromosome counts. BLD was strongly associated with X chromosome count in both male and female groups.

Conclusions

Our results suggest that X- and Y-linked genes have differential effects on CC morphometry. To our knowledge, this is the first study to compare CC morphometry across these extremely rare groups.

     

Molecular phylogeny of the Robust clade (Faviidae, Mussidae, Merulinidae, and Pectiniidae): An Indian Ocean perspective

Recent phylogenetic analyses have demonstrated the limits of traditional coral taxonomy based solely on skeletal morphology. In this phylogenetic context, Faviidae and Mussidae are ecologically dominant families comprising one third of scleractinian reef coral genera, but their phylogenies remain partially unresolved. Many of their taxa are scattered throughout most of the clades of the Robust group, and major systematic incongruences exist. Numerous genera and species remain unstudied, and the entire biogeographic area of the Indian Ocean remains largely unsampled. In this study, we analyzed a portion of the mitochondrial cytochrome c oxidase subunit 1 gene and a portion of ribosomal DNA for 14 genera and 27 species of the Faviidae and Mussidae collected from the Indian Ocean and New Caledonia and this is the first analysis of five of these species. For some taxa, newly discovered evolutionary relationships were detected, such as the evolutionary distinctiveness of Acanthastrea maxima, the genetic overlap of Parasimplastrea omanensis and Blastomussa merleti, and the peculiar position of Favites peresi in clade XVII together with Echinopora and Montastraea salebrosa. Moreover, numerous cases of intraspecific divergences between Indian Ocean and Pacific Ocean populations were detected. The most striking cases involve the genera Favites and Favia, and in particular Favites complanata, F. halicora, Favia favus, F. pallida, F. matthaii, and F. rotumana, but divergence also is evident in Blastomussa merleti, Cyphastrea serailia, and Echinopora gemmacea. High morphological variability characterizes most of these taxa, thus traditional skeletal characteristics, such as corallite arrangement, seem to be evolutionary misleading and are plagued by convergence. Our results indicate that the systematics of the Faviidae and the Mussidae is far from being resolved and that the inclusion of conspecific populations of different geographical origin represents an unavoidable step when redescribing the taxonomy and systematics of scleractinian corals. More molecular phylogenies are needed to define the evolutionary lineages that could be corroborated by known and newly discovered micromorphological characters.     

Oxidative metabolism of dopamine: a colour reaction from human midbrain analysed by mass spectrometry

In order to identify the protein responsible for a dopamine peroxidizing activity, previously described in human normal and parkinsonian substantia nigra by our group, we developed non-denaturing polyacrylamide gel electrophoresis conditions, mimicking the characteristic colour in vitro reaction, resulting from cyclic oxidation of dopamine (DA). After separating protein mixtures from human normal midbrain homogenates on two sets of identical native gels, one gel set was subjected to specific activity staining by using DA and hydrogen peroxide. An activity red/orange band appeared in midbrain tissue lanes, similarly to the lane where commercial horseradish peroxidase (HRP) was present as control of peroxidative activity. The second set of gels, stained with Coomassie Blue, showed other, not enzymatically active protein bands. Mass spectrometry analysis of the bands containing the activity and the corresponding Coomassie Blue bands revealed the presence of proteins that may play a role in neurodegenerative disease, highlighting a possible functional link among dopamine/dopaminochrome redox cycle and protein metabolism.     

Glycolytic enzyme expression and pyruvate kinase activity in cultured fibroblasts from type 1 diabetic patients with and without nephropathy

Since type 1 diabetes mellitus (T1DM) patients with nephropathy (DN+) are insulin-resistant, we aimed to identify (new) potential molecular sites involved in the alterations of glucose metabolism in these patients. We examined the expression of glycolytic enzymes in cultured fibroblasts from T1DM(DN+) patients as compared to those from T1DM patients without nephropathy (DN-) and from controls. Pyruvate kinase (PK) activity was also determined. Human skin fibroblasts were grown in normal glucose (6 mM). RNAs and proteins were analyzed, respectively, using cRNA microarray and two-dimensional electrophoresis followed by identification with mass spectrometry. PK activity was measured using a spectrophotometric assay. As compared to controls, increases in the gene expression of hexokinase, phosphoglucomutase, phosphofructokinase, aldolase and triosephosphate isomerase were found in T1DM(DN+) patients, but not in T1DM(DN-) patients. In T1DM(DN+) patients, the protein analysis showed an altered expression of three glycolytic enzymes: triosophosphate isomerase, enolase and PK. In addition, PK activity in fibroblasts from T1DM(DN+) patients was lower than that in T1DM(DN-) and in controls. In conclusion, this study reports novel alterations of enzymes involved in glucose metabolism that may be associated with the pathophysiology of insulin resistance and of renal damage in T1DM(DN+) patients.     

Exercise therapy for prevention of falls in people with Parkinson's disease: A protocol for a randomised controlled trial and economic evaluation

Background

People with Parkinson's disease are twice as likely to be recurrent fallers compared to other older people. As these falls have devastating consequences, there is an urgent need to identify and test innovative interventions with the potential to reduce falls in people with Parkinson's disease. The main objective of this randomised controlled trial is to determine whether fall rates can be reduced in people with Parkinson's disease using exercise targeting three potentially remediable risk factors for falls (reduced balance, reduced leg muscle strength and freezing of gait). In addition we will establish the cost effectiveness of the exercise program from the health provider's perspective.

Methods/Design

230 community-dwelling participants with idiopathic Parkinson's disease will be recruited. Eligible participants will also have a history of falls or be identified as being at risk of falls on assessment. Participants will be randomly allocated to a usual-care control group or an intervention group which will undertake weight-bearing balance and strengthening exercises and use cueing strategies to address freezing of gait. The intervention group will choose between the home-based or support group-based mode of the program. Participants in both groups will receive standardized falls prevention advice. The primary outcome measure will be fall rates. Participants will record falls and medical interventions in a diary for the duration of the 6-month intervention period. Secondary measures include the Parkinson's Disease Falls Risk Score, maximal leg muscle strength, standing balance, the Short Physical Performance Battery, freezing of gait, health and well being, habitual physical activity and positive and negative affect schedule.

Discussion

No adequately powered studies have investigated exercise interventions aimed at reducing falls in people with Parkinson's disease. This trial will determine the effectiveness of the exercise intervention in reducing falls and its cost effectiveness. This pragmatic program, if found to be effective, has the potential to be implemented within existing community services.

Trial registration

The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12608000303347).