Preparation, biological & cheminformatics-based assessment of N2,N4-diphenylpyrimidine-2,4-diamine as potential Kinase-targeted antimalarials.

Twenty eight new N2,N4-diphenylpyrimidine-2,4-diamines have been prepared in order to expand our understanding of the anti-malarial SAR of the scaffold. The aim of the study was to make structural modifications to improve the overall potency, selectivity and solubility of the series by varying the anilino groups attached to the 2- and 4-position. We evaluated the activity of the compounds against Plasmodium falciparum (Pf) 3D7, cytotoxicity against HepG2, % inhibition at a panel of 10 human kinases, solubility, permeability and lipophilicity, and human and rat in vitro clearance. 11 was identified as a potent anti-malarial with an IC50 of 0.66 µM at the 3D7 strain and a selectivity (SI) of ~ 40 in terms of cytotoxicity against the HepG2 cell line. It also displayed low experimental logD7.4 (2.27), reasonable solubility (124 µg/ml), good metabolic stability, but low permeability. A proteo-chemometric workflow was employed to identify putative Pf targets of the most promising compounds. Ligand-based similarity searching of the ChEMBL database led to the identification of most probable human targets. These were then used as input for sequence-based searching of the Pf proteome. Homology modelling and molecular docking were used to evaluate whether compounds could indeed bind to these targets with valid binding modes. In vitro biological testing against close human analogs of these targets was subsequently undertaken. This allowed us to identify potential Pf targets and human anti-targets that could be exploited in future development.

An assessment study of known pyrazolopyrimidines: Chemical methodology and cellular activity.

Heterocyclic compounds with nitrogen atom play a key role in the normal life cycle of a cell. Pyrazolopyrimidine is a privileged class of nitrogen containing fused heterocyclic compound contributing to a major portion of all lead molecules in medicinal chemistry. The thumbprint of pyrazolopyrimidine as a pharmacophore is always noticeable due to its analogy with the adenine base in DNA. Pyrazolopyrimidines are divided into five types [I, II, III, IV, V] based on the mechanism of action on the specific target conferring a wide scope of research which has accelerated the interest of researchers to investigate its biological profile. In 1956, the anti-cancer activity of pyrazolopyrimidine was evaluated for the first time with appreciable results. Since then, medicinal chemists centered their work on various methods of synthesis and evaluating the biological profile of pyrazolopyrimidine isomers. This report consists of novel methodologies followed to synthesize pyrazolopyrimidine isomers along with a note on their biological significance. To the best of our knowledge, this review article will be first of its kind to encompass different synthetic procedures along with anti-cancer, kinase inhibition, phosphodiesterase inhibition and receptor blocking activity of pyrazolopyrimidine moieties. IC50 values of potent compounds are added wherever necessary to understand the suitability of pyrazolopyrimidine skeletons for a specific biological activity.

Effective reaction rates in diffusion-limited phosphorylation-dephosphorylation cycles.

We investigate the kinetics of the ubiquitous phosphorylation-dephosphorylation cycle on biological membranes by means of kinetic Monte Carlo simulations on the triangular lattice. We establish the dependence of effective macroscopic reaction rate coefficients as well as the steady-state phosphorylated substrate fraction on the diffusion coefficient and concentrations of opposing enzymes: kinases and phosphatases. In the limits of zero and infinite diffusion, the numerical results agree with analytical predictions; these two limits give the lower and the upper bound for the macroscopic rate coefficients, respectively. In the zero-diffusion limit, which is important in the analysis of dense systems, phosphorylation and dephosphorylation reactions can convert only these substrates which remain in contact with opposing enzymes. In the most studied regime of nonzero but small diffusion, a contribution linearly proportional to the diffusion coefficient appears in the reaction rate. In this regime, the presence of opposing enzymes creates inhomogeneities in the (de)phosphorylated substrate distributions: The spatial correlation function shows that enzymes are surrounded by clouds of converted substrates. This effect becomes important at low enzyme concentrations, substantially lowering effective reaction rates. Effective reaction rates decrease with decreasing diffusion and this dependence is more pronounced for the less-abundant enzyme. Consequently, the steady-state fraction of phosphorylated substrates can increase or decrease with diffusion, depending on relative concentrations of both enzymes. Additionally, steady states are controlled by molecular crowders which, mostly by lowering the effective diffusion of reactants, favor the more abundant enzyme.

Systematic assessment of coordinated activity cliffs formed by kinase inhibitors and detailed characterization of activity cliff clusters and associated SAR information.

From currently available kinase inhibitors and their activity data, clusters of coordinated activity cliffs were systematically derived and subjected to cluster index and index map analysis. Type I-like inhibitors with well-defined IC50 measurements were found to provide a large knowledge base of activity cliff clusters for 266 targets from nine kinase groups. On the basis of index map analysis, these clusters were systematically organized according to structural similarity of inhibitors and activity cliff diversity and prioritized for structure-activity relationship (SAR) analysis. From prioritized clusters, interpretable SAR information can be extracted. It is also shown that activity cliff clusters formed by ATP site-directed inhibitors often represent local SAR environments of rather different complexity and interpretability. In addition, activity cliff clusters including promiscuous kinase inhibitors have been determined. Only a small subset of inhibitors was found to change activity cliff roles in different clusters. The activity cliff clusters described herein and their index map organization substantially enrich SAR information associated with kinase inhibitors in compound subsets of limited size. The cluster and index map information is made available upon request to provide opportunities for further SAR exploration. On the basis of our analysis and the data provided, activity cliff clusters and corresponding inhibitor series for kinase targets of interest can be readily selected.

Stochastic transitions in a bistable reaction system on the membrane.

Transitions between steady states of a multi-stable stochastic system in the perfectly mixed chemical reactor are possible only because of stochastic switching. In realistic cellular conditions, where diffusion is limited, transitions between steady states can also follow from the propagation of travelling waves. Here, we study the interplay between the two modes of transition for a prototype bistable system of kinase-phosphatase interactions on the plasma membrane. Within microscopic kinetic Monte Carlo simulations on the hexagonal lattice, we observed that for finite diffusion the behaviour of the spatially extended system differs qualitatively from the behaviour of the same system in the well-mixed regime. Even when a small isolated subcompartment remains mostly inactive, the chemical travelling wave may propagate, leading to the activation of a larger compartment. The activating wave can be induced after a small subdomain is activated as a result of a stochastic fluctuation. Such a spontaneous onset of activity is radically more probable in subdomains characterized by slower diffusion. Our results show that a local immobilization of substrates can lead to the global activation of membrane proteins by the mechanism that involves stochastic fluctuations followed by the propagation of a semi-deterministic travelling wave.

Dynamics of a stochastic spatially extended system predicted by comparing deterministic and stochastic attractors of the corresponding birth-death process.

Living cells may be considered as biochemical reactors of multiple steady states. Transitions between these states are enabled by noise, or, in spatially extended systems, may occur due to the traveling wave propagation. We analyze a one-dimensional bistable stochastic birth-death process by means of potential and temperature fields. The potential is defined by the deterministic limit of the process, while the temperature field is governed by noise. The stable steady state in which the potential has its global minimum defines the global deterministic attractor. For the stochastic system, in the low noise limit, the stationary probability distribution becomes unimodal, concentrated in one of two stable steady states, defined in this study as the global stochastic attractor. Interestingly, these two attractors may be located in different steady states. This observation suggests that the asymptotic behavior of spatially extended stochastic systems depends on the substrate diffusivity and size of the reactor. We confirmed this hypothesis within kinetic Monte Carlo simulations of a bistable reaction- diffusion model on the hexagonal lattice. In particular, we found that although the kinase-phosphatase system remains inactive in a small domain, the activatory traveling wave may propagate when a larger domain is considered.

An information theoretical analysis of kinase activated phosphorylation dephosphorylation cycle.

Signal transduction, the information processing mechanism in biological cells, is carried out by a network of biochemical reactions. The dynamics of driven biochemical reactions can be studied in terms of nonequilibrium statistical physics. Such systems may also be studied in terms of Shannon's information theory. We combine these two perspectives in this study of the basic units (modules) of cellular signaling: the phosphorylation dephosphorylation cycle (PdPC) and the guanosine triphosphatase (GTPase). We show that the channel capacity is zero if and only if the free energy expenditure of biochemical system is zero. In fact, a positive correlation between the channel capacity and free energy expenditure is observed. In terms of the information theory, a linear signaling cascade consisting of multiple steps of PdPC can function as a distributed "multistage code." With increasing number of steps in the cascade, the system trades channel capacity with the code complexity. Our analysis shows that while a static code can be molecular structure based, a biochemical communication channel has to have energy expenditure.

Single-variable reaction systems: deterministic and stochastic models.

Biochemical reaction networks are often described by deterministic models based on macroscopic rate equations. However, for small numbers of molecules, intrinsic noise can play a significant role and stochastic methods may thus be required. In this work, we analyze the differences and similarities between a class of macroscopic deterministic models and corresponding mesoscopic stochastic models. We derive expressions that provide a clear and intuitive view upon the behavior of the stochastic model. In particular, these expressions show the dependence of both the dynamics and the stationary distribution of the stochastic model on the number of molecules in the system. As expected, most properties of the stochastic model correspond well with those in the deterministic model if the number of molecules is large enough. However, for some properties, both models are inconsistent, even if the number of molecules in the stochastic model tends to infinity. Throughout this paper, we use a bistable autophosphorylation cycle as a running example. For such a bistable system, we give an explicit proof that the rate of convergence to the stationary distribution (or the second eigenvalue of the transition matrix) depends exponentially on the number of molecules.

Miniaturisation of a high throughput screening assay comparing air displacement and capillary-based nanolitre transfer technologies.

The miniaturisation of high-throughput screening (HTS) assays has been a widely debated and researched strategy with the aims of reducing screening costs and increasing speed while not compromising data quality. In this context, liquid handling technologies continue to be improved. A new and promising development is the emergence of nanolitre dispensers, which are capable of direct compound transfer to assay microplates. In this study, we investigated the miniaturisation of a HTS kinase assay and compared the real life performance of current state-of-the-art air displacement transfer technology (MiniTrak V System) and a capillary based nanolitre dispenser (CyBi-HummingWell). The robustness and effectiveness of the miniaturised assay formats were compared by testing staurosporine to generate dose-response curves and 340 previously identified active compounds. Using the MiniTrak device, assay miniaturisation was achieved from 18 microL to 6 microL in 384-well and 1536-well plate formats. Utilising the nanolitre dispenser, miniaturisation was performed down to 5 microL in 1536-well plates. The Z' factors obtained for each assay format were consistently above 0.5. The data presented here describe the reproducibility of the results obtained with the two transfer technologies and highlight possible issues for hit identification.

Assessment of chemical coverage of kinome space and its implications for kinase drug discovery.

More than 500 compounds chosen to represent kinase inhibitor space have been screened against a panel of over 200 protein kinases. Significant results include the identification of hits against new kinases including PIM1 and MPSK1, and the expansion of the inhibition profiles of several literature compounds. A detailed analysis of the data through the use of affinity fingerprints has produced findings with implications for biological target selection, the choice of tool compounds for target validation, and lead discovery and optimization. In a detailed examination of the tyrosine kinases, interesting relationships have been found between targets and compounds. Taken together, these results show how broad cross-profiling can provide important insights to assist kinase drug discovery.

How large does a compound screening collection need to be?

Increasingly, chemical libraries are being produced which are focused on a biological target or group of related targets, rather than simply being constructed in a combinatorial fashion. A screening collection compiled from such libraries will contain multiple analogues of a number of discrete series of compounds. The question arises as to how many analogues are necessary to represent each series in order to ensure that an active series will be identified. Based on a simple probabilistic argument and supported by in-house screening data, guidelines are given for the number of compounds necessary to achieve a "hit", or series of hits, at various levels of certainty. Obtaining more than one hit from the same series is useful since this gives early acquisition of SAR (structure-activity relationship) and confirms a hit is not a singleton. We show that screening collections composed of only small numbers of analogues of each series are sub-optimal for SAR acquisition. Based on these studies, we recommend a minimum series size of about 200 compounds. This gives a high probability of confirmatory SAR (i.e. at least two hits from the same series). More substantial early SAR (at least 5 hits from the same series) can be gained by using series of about 650 compounds each. With this level of information being generated, more accurate assessment of the likely success of the series in hit-to-lead and later stage development becomes possible.

Diffusion-limited reactions in G-protein activation: unexpected consequences of antagonist and agonist competition.

In this work, we ask whether the simultaneous movement of agonist and antagonist among surface receptors (i.e. continually associating and dissociating from individual receptors according to specified kinetics) has any unexpected consequences for G-protein activation and receptor desensitization. A Monte Carlo model framework is used to track the diffusion and reaction of individual receptors, allowing the requirement for receptors and G-proteins or receptors and kinases to find each other by diffusion (collision coupling) to be implemented explicitly. We find that at constant agonist occupancy the effect of an antagonist on both G-protein activation and the ratio of G-protein activation to receptor desensitization can be modulated by varying the antagonist dissociation kinetics. The explanation for this effect is that antagonist dissociation kinetics influence the ability of agonists to access particular receptors and thus reach G-proteins and kinases near those receptors. Relevant parameter ranges for observation of these effects are identified. These results are useful for understanding experimental and therapeutic situations when both agonist and antagonist are present, and in addition may offer new insights into insurmountable antagonism.

Small-molecule kinase-inhibitor target assessment.

The identification of the cellular targets of small-molecule protein kinase inhibitors is a significant hurdle to assessing their therapeutic potential for many diseases. Here we review several biochemical and genetics-based approaches to identifying inhibitor targets. We also describe a chemical-genomics approach to kinase-inhibitor target identification and validation that matches transcriptional signatures elicited by a drug of unknown specificity and those elicited by highly specific pharmacological inhibition of engineered candidate kinase targets.

Experiences in implementing uHTS--cutting edge technology meets the real world.

Driven by growing corporate compound files, the demands of target biology, and attempts to cut cost, the number of solutions to HTS has spiralled. In quick succession new assay technologies and screening platforms are appearing on the market, with the promise of screening faster than ever in low volume high density formats whilst providing high quality data. Within this world of rapid change, Pfizer has applied cutting edge technology to HTS by introducing screening in 1 microl formats utilising single molecule detection technology. Instead of resource intensive in-house development, Pfizer entered into a collaboration with Evotec OAI / Evotec Technologies and introduced their Mark-II EVOscreen platform. In this article we will outline the benefits of the approach taken at Pfizer, Sandwich, and introduce the Mark-II EVOscreen platform, illustrating the potential but also possible pitfalls of HTS miniaturisation.

Morphological assessment of the development of multinucleated giant cells in glioma by using mitosis-specific phosphorylated antibodies.

OBJECT: The nature and origin of multinucleated giant cells in glioma have not been made clear. To investigate the phosphorylation of intermediate filaments, the authors studied multinucleated giant cells in vitro and in vivo by using mitosis-specific phosphorylated antibodies. METHODS: Cultured human glioma cells were immunostained with monoclonal antibodies (mAbs) 4A4, KT13, and TM71, which recognized the phosphorylation of vimentin at Ser55, glial fibrillary acidic protein at Serl3, and vimentin at Ser71, respectively. Subsequently, the nature of multinucleated giant cells was investigated using laser scanning confocal microscopy. In addition, paraffin-embedded tissue sections obtained in three patients with giant cell glioblastoma were also investigated. Multinucleated giant cells were immunoreacted with the mAb 4A4 and not with KT13 and TM71 in vitro and in vivo. In addition, the authors obtained these results in multinucleated giant cells under natural conditions, without drug treatments. CONCLUSIONS: Findings in this investigation indicated that multinucleated giant cells are those remaining in mitosis between metaphase and telophase, undergoing neither fusion nor degeneration.

Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement.

Efficient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1-catalyzed ss-phosphoryl transfer in mice decreases the potential of myofibers to sustain nucleotide ratios despite up-regulation of high-energy phosphoryl flux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1-deficient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1(-/-) muscles. Thus, AK1-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost.

In vivo and in vitro assessment of mitogen activated protein kinase involvement during quail secondary palate formation.

Spatiotemporally regulated cell proliferation and differentiation are crucial for the successful completion of morphogenesis of the vertebrate secondary palate. An understanding of the mechanisms by which these cellular phenomena are regulated during palate development involves the identification of the various signal transduction pathways. In the present study, the presence and activation of mitogen-activated protein (MAP) kinases were investigated during the development of quail secondary palate. The palatal shelves were dissected on days 5-9 of incubation, homogenized, and centrifuged, after which the samples were separated by anion exchange fast protein liquid chromatography. The fractions were analyzed for myelin basic protein (MBP) phosphorylation. In addition, primary cultures of quail palate mesenchymal cells (QPMCs) were treated with epidermal growth factor (EGF) and prepared for MBP phosphorylation assays. A temporally regulated pattern of phosphotransferase activity, characterized by a three-fold increase in phosphotransferase activity toward MBP between days 5 and 8 of incubation, was observed during quail palate development. Western blotting, using MAP kinase antibodies, demonstrated the presence of a 42-kDa isoform between days 5 and 9 of incubation, during which the level of protein remained constant. Antityrosine immunoblotting with 4G10 also detected a 42-kDa protein. Phosphotransferase assays, using either a MAP kinase-specific substrate peptide (S5) or a protein kinase C inhibitor (R3), further confirmed the presence of a MAP kinase in the developing palate of quail. Because diverse biological processes occur concurrently during in vivo palate morphogenesis, the involvement of MAP kinase was explored further in primary cell culture. The data showed that EGF stimulated proliferation and activated 42-kDa MAP kinase in QPMCs. It is suggested that MAP kinase cascade may be involved in growth factor-regulated cell proliferation during morphogenesis of quail secondary palate.

Organization of the phosphoinositide cycle. Assessment of inositol transferase activity in purified plasma membranes.

Experiments were carried out to determine whether or not CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase (IT) activity (EC 2.7.8.11) could be detected in purified plasma-membrane fractions from WRK-1 rat mammary tumour cells. These cells have previously been shown to have a very active phosphoinositide cycle. Sucrose-density-gradient-purified plasma membranes contained no IT activity that could not be accounted for by endoplasmic-reticulum contamination. However, we also determined that the relative amount of IT activity in endoplasmic reticulum and plasma-membrane fractions could be altered by changing the concentration of detergent in the assay system.