Dissecting isoform selectivity of PI3K inhibitors: the role of non-conserved residues in the catalytic pocket

The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr(886) and Lys(890) of the PI3Kgamma isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the beta isoform (Glu(858) and Asp(862)) and those in the alpha isoform (His(855) and Gln(859)) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of beta-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the alpha-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kbeta activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the alpha-isoform-selectivity of the compounds studied.     

A novel antioxidant agent caffeic acid phenethyl ester prevents long-term mobile phone exposure-induced renal impairment in rat

Caffeic acid phenethyl ester (CAPE), a flavonoid like compound, is one of the major components of honeybee propolis. It has been used in folk medicine for many years in Middle East countries. It was found to be a potent free radical scavenger and antioxidant recently. The aim of this study was to examine long-term applied 900 MHz emitting mobile phone-induced oxidative stress that promotes production of reactive oxygen species (ROS) and, was to investigate the role of CAPE on kidney tissue against the possible electromagnetic radiation (EMR)-induced renal impairment in rats. In particular, the ROS such as superoxide and nitric oxide (NO) may contribute to the pathophysiology of EMR-induced renal impairment. Malondialdehyde (MDA, an index of lipid peroxidation) levels, urinary N-acetyl-β-d-glucosaminidase (NAG, a marker of renal tubular injury) and nitric oxide (NO, an oxidant product) levels were used as markers of oxidative stress-induced renal impairment and the success of CAPE treatment. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in renal tissue were determined to evaluate the changes of antioxidant status. The rats used in the study were randomly grouped (10 each) as follows: i) Control group (without stress and EMR), ii) Sham-operated rats stayed without exposure to EMR (exposure device off), iii) Rats exposed to 900 MHz EMR (EMR group), and iv) A 900 MHz EMR exposed + CAPE treated group (EMR + CAPE group). In the EMR exposed group, while tissue MDA, NO levels and urinary NAG levels increased (p < 0.0001), the activities of SOD, CAT, and GSH-Px in renal tissue were reduced (p < 0.001). CAPE treatment reversed these effects as well (p < 0.0001, p < 0.001 respectively). In conclusion, the increase in NO and MDA levels of renal tissue, and in urinary NAG with the decrease in renal SOD, CAT, GSH-Px activities demonstrate the role of oxidative mechanisms in 900 MHz mobile phone-induced renal tissue damage, and CAPE, via its free radical scavenging and antioxidant properties, ameliorates oxidative renal damage. These results strongly suggest that CAPE exhibits a protective effect on mobile phone-induced and free radical mediated oxidative renal impairment in rats.     

Predicting the grass pollen count from meteorological data with regard to estimating the severity of hayfever symptoms in Melbourne (Australia)

In Melbourne, Australia, grass pollen is the predominant cause of hayfever in late spring and summer. The grass pollen season has been monitored in Melbourne, using a Burkard spore trap, for 13 years (1975–1981, 1985 and 1991–1997). Total counts for grass pollen were highly variable from one season to the next (approximately 1000 to >8000 grains/m³). The daily grass pollen counts also showed a high variability (0 to approximately 400 grains/m³). In this study, the grass pollen counts of the 13 years (12 grass pollen seasons, extending from October to January) have been compared with meteorological data in order to identify the conditions that can determine the daily amounts of grass pollen in the air. It was found that the seasonal total of grass pollen was directly correlated with the rainfall sum of the preceding 12 months (1 September–31 August): seasonal total of grass pollen (counts/m³)=18.161 × rainfall sum of the preceding 12 months (mm) −8541.5 (r _s=0.74,P<0.005,n=12). The daily amounts of grass pollen in the air were positively correlated with the corresponding daily average ambient temperatures (P<0.001). The daily amount of grass pollen which was to be expected with a certain daily average temperature was linked to the seasonal total of grass pollen: in years with high total grass pollen counts, a lower daily average temperature was required for a high daily pollen count than in years with low total grass pollen counts. As the concentration of airborne grass pollen determines the severity of hayfever in sensitive patients, an estimation of daily grass pollen counts can provide an indication of potential pollinosis symptoms. We compared daily grass pollen counts with the reported symptomatic responses of hayfever sufferers in November 1985 and found that hayfever symptoms were significantly correlated to the grass pollen counts (P<0.001 for nasal,P<0.005 for eye symptoms). Thus, a combination of meteorological information (i.e. rainfall and temperature) allows for an estimation of the potential daily pollinosis symptoms during the grass pollen season. Here we propose a symptom estimation chart, allowing a quick prediction of eye and nasal symptoms that are likely to occur as a result of variations in meteorological conditions, thus enabling both physicians and patients to take appropriate avoidance measures or therapy.     

Machine learning-based model predictive controller design for cell culture processes

The biopharmaceutical industry continuously seeks to optimize the critical quality attributes to maintain the reliability and cost-effectiveness of its products. Such optimization demands a scalable and optimal control strategy to meet the process constraints and objectives. This work uses a model predictive controller (MPC) to compute an optimal feeding strategy leading to maximized cell growth and metabolite production in fed-batch cell culture processes. The lack of high-fidelity physics-based models and the high complexity of cell culture processes motivated us to use machine learning algorithms in the forecast model to aid our development. We took advantage of linear regression, the Gaussian process and neural network models in the MPC design to maximize the daily protein production for each batch. The control scheme of the cell culture process solves an optimization problem while maintaining all metabolites and cell culture process variables within the specification. The linear and nonlinear models are developed based on real cell culture process data, and the performance of the designed controllers is evaluated by running several real-time experiments.     

Predicting the binding properties of cibacron blue F3GA in affinity separation systems

The binding properties of cibacron blue F3GA (CB-F3GA) bound to a model NAD(P)H/FAD(H2)-dependent protein system, namely cytosolic quinone reductase (QR), was characterized by AMBER in an attempt to address the binding properties of immobilized CB-F3GA used in the separation of serum albumin. A favorable binding free energy of -4.52kcal/mol (KD=5.09 x 10(-4)kcal/mol) was determined for CB-F3GA binding by MM-PBSA method, which was found to be a ballpark estimate of empirical values reported in literature (DeltaG approximately -6kcal/mol). We propose that CB-F3GA primarily follows a class III binding motif in presence of FAD in the binding site of QR in solution, while a class II binding motif is observed in the crystal form. It was found that favorable van der Waals/hydrophobic interactions take place in the binding site making a major contribution to a favorably dominating enthalpy of binding (DeltaHtot=-25.87kcal/mol) as compared to a disfavorable binding entropy term (TDeltaStot=-21.35kcal/mol). Additional MM-PBSA experiments in the absence of FAD gave rise to a disfavorable binding free energy for CB in complex with QR, suggesting that FAD is an essential determinant of CB-F3GA binding. This is in contrast to an earlier observation of Denizli et al. on separation of human serum albumin (HSA) by immobilized CB-F3GA in the absence of FAD. Therefore, a class I binding model for CB-F3GA is proposed here to account for the efficient separation of HSA in affinity chromatography systems.     

Maternal Embryonic Leucine Zipper Kinase (MELK) Reduces Replication Stress in Glioblastoma Cells

Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G₁/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21^WAF1/CIP1, an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies.     

The Sirtuin-2 Inhibitor AK7 Is Neuroprotective in Models of Parkinson’s Disease but Not Amyotrophic Lateral Sclerosis and Cerebral Ischemia

Sirtuin deacetylases regulate diverse cellular pathways and influence disease processes. Our previous studies identified the brain-enriched sirtuin-2 (SIRT2) deacetylase as a potential drug target to counteract neurodegeneration. In the present study, we characterize SIRT2 inhibition activity of the brain-permeable compound AK7 and examine the efficacy of this small molecule in models of Parkinson’s disease, amyotrophic lateral sclerosis and cerebral ischemia. Our results demonstrate that AK7 is neuroprotective in models of Parkinson’s disease; it ameliorates alpha-synuclein toxicity in vitro and prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion and dopaminergic neuron loss in vivo. The compound does not show beneficial effects in mouse models of amyotrophic lateral sclerosis and cerebral ischemia. These findings underscore the specificity of protective effects observed here in models of Parkinson’s disease, and previously in Huntington’s disease, and support the development of SIRT2 inhibitors as potential therapeutics for the two neurodegenerative diseases.