Predicting P-Glycoprotein-Mediated Drug Transport Based On Support Vector Machine and Three-Dimensional Crystal Structure of P-glycoprotein

Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening

Bcl-2 protein family: Implications in vascular apoptosis and atherosclerosis

Apoptosis has been recognized as a central component in the pathogenesis of atherosclerosis, in addition to the other human pathologies such as cancer and diabetes. The pathophysiology of atherosclerosis is complex, involving both apoptosis and proliferation at different phases of its progression. Oxidative modification of lipids and inflammation differentially regulate the apoptotic and proliferative responses of vascular cells during progression of the atherosclerotic lesion. Bcl-2 proteins act as the major regulators of extrinsic and intrinsic apoptosis signalling pathways and more recently it has become evident that they mediate the apoptotic response of vascular cells in response to oxidation and inflammation either in a provocative or an inhibitory mode of action. Here we address Bcl-2 proteins as major therapeutic targets for the treatment of atherosclerosis and underscore the need for the novel preventive and therapeutic interventions against atherosclerosis, which should be designed in the light of molecular mechanisms regulating apoptosis of vascular cells in atherosclerotic lesions

Applying refinement to the use of mice and rats in rheumatoid arthritis research

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat ‘models’ of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research

Effectiveness of joint mobilisation after cast immobilisation for ankle fracture: a protocol for a randomised controlled trial [ACTRN012605000143628]

BACKGROUND: Passive joint mobilisation is a technique frequently used by physiotherapists to reduce pain, improve joint movement and facilitate a return to activities after injury, but its use after ankle fracture is currently based on limited evidence. The primary aim of this trial is to determine if adding joint mobilisation to a standard exercise programme is effective and cost-effective after cast immobilisation for ankle fracture in adults. METHODS/DESIGN: Ninety participants will be recruited from the physiotherapy departments of three teaching hospitals and randomly allocated to treatment or control groups using a concealed procedure. All participants will perform an exercise programme. Participants in the treatment group will also receive joint mobilisation twice a week for four weeks. Blinded follow-up assessments will be conducted four, 12 and 24 weeks after randomisation. The primary outcome measures will be the Lower Extremity Functional Scale and the Assessment of Quality of Life. Secondary outcomes will include measures of impairments, activity limitation and participation. Data on the use of physiotherapy services and participants' out-of-pocket costs will be collected for the cost-effective and cost-utility analyses. To test the effects of treatment, between-group differences will be examined with analysis of covariance using a regression approach. The primary conclusions will be based on the four-week follow-up data. DISCUSSION: This trial incorporates features known to minimise bias. It uses a pragmatic design to reflect clinical practice and maximise generalisability. Results from this trial will contribute to an evidence-based approach for rehabilitation after ankle fracture

Novel Mitochondrial Substrates of Omi Indicate a New Regulatory Role in Neurodegenerative Disorders

The mitochondrial protease OMI (also known as HtrA2) has been implicated in Parkinson's Disease (PD) and deletion or protease domain point mutations have shown profound neuropathologies in mice. A beneficial role by OMI, in preserving cell viability, is assumed to occur via the avoidance of dysfunctional protein turnover. However relatively few substrates for mitochondrial Omi are known. Here we report our identification of three novel mitochondrial substrates that impact metabolism and ATP production. Using a dual proteomic approach we have identified three interactors based upon ability to bind to OMI, and/or to persist in the proteome after OMI activity has been selectively inhibited. One candidate, the chaperone HSPA8, was common to each independent study. Two others (PDHB subunit and IDH3A subunit) did not appear to bind to OMI, however persisted in the mito-proteome when OMI was inhibited. Pyruvate dehydrogenase (PDH) and isocitrate dehydrogenase (IDH) are two key Kreb's cycle enzymes that catalyse oxidative decarboxylation control points in mitochondrial respiration. We verified both PDHB and IDH3A co-immunoprecipitate with HSPA8 and after elution, were degraded by recombinant HtrA2 in vitro. Additionally our gene expression studies, using rotenone (an inhibitor of Complex I) showed Omi expression was silenced when pdhb and idh3a were increased when a sub-lethal dose was applied. However higher dose treatment caused increased Omi expression and decreased levels of pdhb and idh3a transcripts. This implicates mitochondrial OMI in a novel mechanism relating to metabolism

Skeletal Muscle Differentiation Evokes Endogenous XIAP to Restrict the Apoptotic Pathway

Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells) are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells) and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP). Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms

Exploiting evolutionary steering to induce collateral drug sensitivity in cancer

Drug resistance mediated by clonal evolution is arguably the biggest problem in cancer therapy today. However, evolving resistance to one drug may come at a cost of decreased fecundity or increased sensitivity to another drug. These evolutionary trade-offs can be exploited using 'evolutionary steering' to control the tumour population and delay resistance. However, recapitulating cancer evolutionary dynamics experimentally remains challenging. Here, we present an approach for evolutionary steering based on a combination of single-cell barcoding, large populations of 108-109 cells grown without re-plating, longitudinal non-destructive monitoring of cancer clones, and mathematical modelling of tumour evolution. We demonstrate evolutionary steering in a lung cancer model, showing that it shifts the clonal composition of the tumour in our favour, leading to collateral sensitivity and proliferative costs. Genomic profiling revealed some of the mechanisms that drive evolved sensitivity. This approach allows modelling evolutionary steering strategies that can potentially control treatment resistance

Loss of p53 in quaking viable mice leads to Purkinje cell defects and reduced survival

The qkv mutation is a one megabase deletion resulting in abnormal expression of the qkI gene. qkv mice exhibit hypomyelination of the central nervous system and display rapid tremors and seizures as adults. The qkI locus on 6q26-27 has also been implicated as a candidate tumor suppressor gene as the qkI locus maps to a region of genetic instability in Glioblastoma Multiforme (GBM), an aggressive brain tumor of astrocytic lineage. As GBM frequently harbors mutations affecting p53, we crossbred qkv and p53 mutant mice to examine whether qkv mice on a p53−/− background have an increased incidence of GBM. qkv/v; p53−/− mice had a reduced survival rate compared to p53−/− littermates, and the cause of death of the majority of the mice remains unknown. In addition, immunohistochemistry revealed Purkinje cell degeneration in the cerebellum. These results suggest that p53 and qkI are genetically linked for neuronal maintenance and survival