Identification of Pak1 inhibitors using water thermodynamic analysis

Abstract

p21-activated kinases (Paks) play an integral component in various cellular diverse processes. The full activation of Pak is dependent upon several serine residues present in the N-terminal region, a threonine present at the activation loop, and finally the phosphorylation of these residues ensure the complete activation of Pak1. The present study deals with the identification of novel potent candidates of Pak1 using computational methods as anti-cancer compounds. A diverse energy based pharmacophore (e-pharmacophore) was developed using four co-crystal inhibitors of Pak1 having pharmacophore features of 5 (DRDRR), 6 (DRHADR), and 7 (RRARDRP and DRRDADH) hypotheses. These models were used for rigorous screening against e-molecule database. The obtained hits were filtered using ADME/T and molecular docking to identify the high affinity binders. These hits were subjected to hierarchical clustering using dendritic fingerprint inorder to identify structurally diverse molecules. The diverse hits were scored against generated water maps to obtain WM/MM ΔG binding energy. Furthermore, molecular dynamics simulation and density functional theory calculations were performed on the final hits to understand the stability of the complexes. Five structurally diverse novel Pak1 inhibitors (4835785, 32198676, 32407813, 76038049, and 32945545) were obtained from virtual screening, water thermodynamics and WM/MM ΔG binding energy. All hits revealed similar mode of binding pattern with the hinge region residues replacing the unstable water molecules in the binding site. The obtained novel hits could be used as a platform to design potent drugs that could be experimentally tested against cancer patients having increased Pak1 expression.     

The role of leaf surface wetness in larval behaviour of the sorghum shoot fly, Atherigona soccata

The susceptibility of sorghum to the shoot fly Atherigona soccata Rondani, (Diptera: Muscidae) is affected by seedling age and is highest when seedlings are 8–12 days old. This corresponds with high moisture accumulation on the central leaf which is the path of newly hatched larva as it moves downwards from the oviposition site, towards the growing apex. Studies showed that leaf surface wetness (LSW) of the central shoot leaf was higher in 10-day old seedlings than in seedlings of other ages. Similarly, LSW was much higher in the susceptible sorghum genotype CSH 1 than in the resistant genotype IS 2146. Larvae moved faster towards the growing point and produced deadhearts much earlier in CSH 1 than in IS 2146. They also moved faster in 10-day old seedlings than in seedlings of other ages. It was also shown that the leaf surface wetness of the central shoot leaf is a more reliable parameter of resistance than the glossy leaf trait or trichome density.

---

L'influence de la humidité de la surface foliaire sur le comportement de la mouche des pousses du sorgho

Résumé La sensibilité du sorgho à la mouche des pousses du sorgho, Atherigona soccata Rondani, est liée à l'âge de la plantule. Elle est plus forte lorsque la plantule est âgée de 8 à 12 jours et la sensibilité est maximale à 10 jours. A ce stade de croissance on observe une forte accumulation d'humidité sur la feuille centrale de la tige. Les jeunes larves traversent cette zone humide lorsqu'elles descendent vers la zone de croissance à partir des pontes déposées sur la face ventrale des feuilles déroulées.Des études ont été menées à l'ICRISAT (Inde) sur la relation entre l'humidité de la feuille centrale de la tige des plantules du sorgho et les dégâts provoqués par la mouche des pousses. L'humidité de la surface des feuilles (HSF) a été estimée grâce à une échelle visuelle graduée 1 à 5 où, 1 = pas d'humidité apparente et 5 = surface de la feuille recouverte de gouttes d'eau. La HSF est plus élevée sur des pousses de sorgho âgées de 10 j que sur les pousses appartenant à d'autres classes d'âge. Les valeurs observées sont également plus fortes pour les variétés non résistantes à ce ravageur (CSH 1,4.8) que pour les variétés résistantes (IS 2146, (2)). La vitesse du déplacement larvaire entre le cornet et la zone de la croissance varie en fonction de l'âge de la plante et des cultivars. Les larves migrent plus rapidement vers la zone de croissance et provoquent la mort du coeur du sorgho plus tôt dans la variété CSH 1 que dans IS 2146. Les larves se déplacent plus rapidement dans les pousses âgées de 10 j que dans les pousses appartenant à d'autres classes d'âge.Des études ont également démontré que la HSF n'est pas directement liée au caractère feuille lisse où à la densité des trichomes. La HSF est faible pour les génotypes résistants présentent où non le caractère feuille lisse. Par contre la HSF est élevée pour les génotypes non résistants présentant le caractère feuille lisse ou non. Aucune relation directe entre la densité des trichomes et les dégâts provoqués par la mouche des pousses n'a pu être mise en évidence. L'analyse des correlations établie pour les caractères de surface des feuilles avec la mort du cur des sorghos indique que les correlations sont faibles et non-significatives pour le caractère feuille lisse (0.49) et la densité des trichomes (0.39 et 0.2). Par contre les correlations sont fortes et significatives pour la HSF (0.82).On conclue que la HSF de la feuille centrale de la tige est un facteur important dans le déterminisme de la résistance du sorgho vis à vis de la mouche des pousses. Les relations entre les processus physiologiques de la plante et les facteurs impliquées dans l'accumulation d'eau sur la surface des feuilles font actuellement l'objet d'études détaillées.

     

Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.     

Changes in UsnRNA biosynthesis during rat liver regeneration

Partial hepatectomy (P.H.) induces a partially synchronized growth response of liver under normal regulation of growth. In this phase changes in cellular morphology, radial distribution pattern of cells and other biological as well as major biochemical changes are well documented [24]. Here, we have shown that the cellular content of UsnRNAs altered during this proliferative phase as well. The level of spliceosomal UsnRNAs (U1, U2, U4–U6) gradually decreased by 30–50% upto 48 hrs of P.H. followed by gradual increase to reach the normal level within one month of P.H. The U3 snRNA level on the other hand, was nearly equal to that in normal liver at 48 hrs of P.H. but in 24 and 72 hrs of P.H. its level was high (4 fold) in contrast to that in other UsnRNAs. Thus, it is clear from our data that the level of all the six UsnRNAs decreased during 48 hrs of P.H. compared to that after first 24 hrs. This has been correlated in the kinetics of UsnRNAs' synthesis (in terms of labelling) in isolated hepatocytes, where the rate of labelling of all the six UsnRNAs increased 20–30% in 24 hrs regenerating hepatocytes (R.H.) followed by sharp decrease by 30–50% within next 24 hrs, compared to that in the normal hepatocytes. But from 72 hrs onwards in R.H. the rate of labelling of all the six UsnRNAs again increased by 30–50% (compared to that in normal hepatocytes) followed by decrease of their labelling-rate to reach the normal level in R.H. within one month of P.H. Thus, it may be concluded that the changes in UsnRNAs' level during the proliferative phase of liver regeneration may be either due to the alteration in the rate of synthesis (in terms of labelling) or along with it differential turn over rate; this phenomenon may have some consequences with the regenerative process of liver.This paper was published in Molecular and Cellular Biochemistry131:67–73, 1994. Kluwer Academic Publishers regret the publication of the only partly corrected version.     

Repeatability of Cerebral Perfusion Using Dynamic Susceptibility Contrast MRI in Glioblastoma Patients

OBJECTIVES

This study evaluates the repeatability of brain perfusion using dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) with a variety of post-processing methods.

METHODS

Thirty-two patients with newly diagnosed glioblastoma were recruited. On a 3-T MRI using a dual-echo, gradient-echo spin-echo DSC-MRI protocol, the patients were scanned twice 1 to 5 days apart. Perfusion maps including cerebral blood volume (CBV) and cerebral blood flow (CBF) were generated using two contrast agent leakage correction methods, along with testing normalization to reference tissue, and application of arterial input function (AIF). Repeatability of CBV and CBF within tumor regions and healthy tissues, identified by structural images, was assessed with intra-class correlation coefficients (ICCs) and repeatability coefficients (RCs). Coefficients of variation (CVs) were reported for selected methods.

RESULTS

CBV and CBF were highly repeatable within tumor with ICC values up to 0.97. However, both CBV and CBF showed lower ICCs for healthy cortical tissues (up to 0.83), healthy gray matter (up to 0.95), and healthy white matter (WM; up to 0.93). The values of CV ranged from 6% to 10% in tumor and 3% to 11% in healthy tissues. The values of RC relative to the mean value of measurement within healthy WM ranged from 22% to 42% in tumor and 7% to 43% in healthy tissues. These percentages show how much variation in perfusion parameter, relative to that in healthy WM, we expect to observe to consider it statistically significant. We also found that normalization improved repeatability, but AIF deconvolution did not.

CONCLUSIONS

DSC-MRI is highly repeatable in high-grade glioma patients.     

Psychoactive pollution suppresses individual differences in fish behaviour

Environmental contamination by pharmaceuticals is global, substantially altering crucial behaviours in animals and impacting on their reproduction and survival. A key question is whether the consequences of these pollutants extend beyond mean behavioural changes, restraining differences in behaviour between individuals. In a controlled, two-year, multigenerational experiment with independent mesocosm populations, we exposed guppies (Poecilia reticulata) to environmentally realistic levels of the ubiquitous pollutant fluoxetine (Prozac). Fish (unexposed: n = 59, low fluoxetine: n = 57, high fluoxetine: n = 58) were repeatedly assayed on four separate occasions for activity and risk-taking behaviour. Fluoxetine homogenized individuals'' activity, with individual variation in populations exposed to even low concentrations falling to less than half that in unexposed populations. To understand the proximate mechanism underlying these changes, we tested the relative contribution of variation within and between individuals to the overall decline in individual variation. We found strong evidence that fluoxetine erodes variation in activity between but not within individuals, revealing the hidden consequences of a ubiquitous contaminant on phenotypic variation in fish—likely to impair adaptive potential to environmental change.     

Pharmacological analysis of CCK2 receptors up-regulated using engineered transcription factors

Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA50 = 9.05+/-0.11, pentagastrin pA50 = 9.11+/-0.13). In addition, binding studies were conducted using [125I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK(D) = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.