Integrating electrochemical, biological, physical, and thermochemical process units to expand the applicability of anaerobic digestion.

Anaerobic digestion (AD) is a mature biotechnology-production platform with millions of installations at homes, farms, and industrial/municipal settings. Large-scale industrial, agricultural, and municipal waste-treatment systems may observe novel integration with electrochemical, biological, physical, and thermochemical process units to make AD more attractive. Without governmental subsidies, AD has often only a relatively low economic return or none at all. Diversification of products besides methane in biogas may help to change this. Here, several sections discuss different process units to: 1) upgrade biogas into biomethane; 2) convert carbon dioxide in biogas to more biomethane; 3) generate cooling power from process heat; 4) produce bio-crude oil (bio-oil) from organic matter; and 5) produce a liquid biochemical product from organic matter. This is not meant to be an exhaustive list, but rather a selection of particularly promising process units from a technological view, which are already integrated with AD or close to full-scale integration.

Efficient hit-finding approaches for histone methyltransferases: the key parameters.

For many novel epigenetics targets the chemical ligand space and structural information were limited until recently and are still largely unknown for some targets. Hit-finding campaigns are therefore dependent on large and chemically diverse libraries. In the specific case of the histone methyltransferase G9a, the authors have been able to apply an efficient process of intelligent selection of compounds for primary screening, rather than screening the full diverse deck of 900 000 compounds to identify hit compounds. A number of different virtual screening methods have been applied for the compound selection, and the results have been analyzed in the context of their individual success rates. For the primary screening of 2112 compounds, a FlashPlate assay format and full-length histone H3.1 substrate were employed. Validation of hit compounds was performed using the orthogonal fluorescence lifetime technology. Rated by purity and IC(50) value, 18 compounds (0.9% of compound screening deck) were finally considered validated primary G9a hits. The hit-finding approach has led to novel chemotypes being identified, which can facilitate hit-to-lead projects. This study demonstrates the power of virtual screening technologies for novel, therapeutically relevant epigenetics protein targets.

A comparative study of fragment screening methods on the p38α kinase: new methods, new insights.

The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.

Fragment-based lead generation: identification of seed fragments by a highly efficient fragment screening technology.

For the detection of the precise and unambiguous binding of fragments to a specific binding site on the target protein, we have developed a novel reporter displacement binding assay technology. The application of this technology for the fragment screening as well as the fragment evolution process with a specific modelling based design strategy is demonstrated for inhibitors of the protein kinase p38alpha. In a fragment screening approach seed fragments were identified which were then used to build compounds from the deep-pocket towards the hinge binding area of the protein kinase p38alpha based on a modelling approach. BIRB796 was used as a blueprint for the alignment of the fragments. The fragment evolution of these deep-pocket binding fragments towards the fully optimized inhibitor BIRB796 included the modulation of the residence time as well as the affinity. The goal of our study was to evaluate the robustness and efficiency of our novel fragment screening technology at high fragment concentrations, compare the screening data with biochemical activity data and to demonstrate the evolution of the hit fragments with fast kinetics, into slow kinetic inhibitors in an in silico approach.

A novel drug discovery concept for tuberculosis: inhibition of bacterial and host cell signalling.

The Mycobacterium tuberculosis genome encodes for eleven eukaryotic-like Ser/Thr protein kinases. At least three of these (PknA, PknB and PknG) are essential for bacterial growth and survival. PknG is secreted by pathogenic mycobacteria, in macrophages to intervene with host cell signalling pathways and to block the fusion of the lysosomes with the phagosome by a still unknown mechanism. Based on our previously published results, we have initiated a drug discovery program, aiming to improve the potency against PknG and the physiochemical properties of the initially identified hit compound, AX20017, from the class of the tetrahydrobenzothiophenes. We have established a radioactive biochemical PknG kinase assay to test the novel analogues around AX20017. We have developed lead molecules with IC50 values in nanomolar range, and demonstrated their antituberculotic effects on human macrophages. Selected leads might ultimately serve the purpose of inducing phagosomal-lysosomal fusion and therefore destroy the residence of the intracellular mycobacteria. It is unclear at this time if these "homeless" mycobacteria are getting killed by the host, but they will be at least vulnerable to the activity of antimycobacterial agents. Released mycobacteria rely on the essential function of PknB for survival, which is our second molecular kinase target. PknB is a transmembrane protein, responsible for the cell growth and morphology. We have screened our library and synthesized novel compounds for the inhibition of PknB. A pharmacophore model was built and 70,000 molecules from our synthesizable virtual library have been screened to identify novel inhibitor scaffolds for the generation of templated compound libraries. Currently, we are using a radioactive kinase assay employing GarA as the putative, physiological substrate of PknB kinase. We have identified hits and generated optimised hit compounds with IC50 values for the inhibition of PknB in the nanomolar range. Yet those promising hits are not potent enough to yield meaningful "minimum inhibitory concentrations" in mycobacterial growth assays. In the course of our future work, we will increase the potency of the next generation of PknB inhibitors in order to improve their antibacterial activity.

An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro.

G-protein-coupled receptors are desensitized by a two-step process. In a first step, G-protein-coupled receptor kinases (GRKs) phosphorylate agonist-activated receptors that subsequently bind to a second class of proteins, the arrestins. GRKs can be classified into three subfamilies, which have been implicated in various diseases. The physiological role(s) of GRKs have been difficult to study as selective inhibitors are not available. We have used SELEX (systematic evolution of ligands by exponential enrichment) to develop RNA aptamers that potently and selectively inhibit GRK2. This process has yielded an aptamer, C13, which bound to GRK2 with a high affinity and inhibited GRK2-catalyzed rhodopsin phosphorylation with an IC50 of 4.1 nM. Phosphorylation of rhodopsin catalyzed by GRK5 was also inhibited, albeit with 20-fold lower potency (IC50 of 79 nM). Furthermore, C13 reveals significant specificity, since almost no inhibitory activity was detectable testing it against a panel of 14 other kinases. The aptamer is two orders of magnitude more potent than the best GRK2 inhibitors described previously and shows high selectivity for the GRK family of protein kinases.

Drug discovery in the kinase inhibitory field using the Nested Chemical Library technology.

Kinase inhibitors are at the forefront of modern drug research, where mostly three technologies are used for hit-and-lead finding: high throughput screening of random libraries, three-dimensional structure-based drug design based on X-ray data, and focused libraries around limited number of new cores. Our novel Nested Chemical Library (NCL) (Vichem Chemie Research Ltd., Budapest, Hungary) technology is based on a knowledge base approach, where focused libraries around selected cores are used to generate pharmacophore models. NCL was designed on the platform of a diverse kinase inhibitory library organized around 97 core structures. We have established a unique, proprietary kinase inhibitory chemistry around these core structures with small focused sublibraries around each core. All the compounds in our NCL library are stored in a big unified Structured Query Language database along with their measured and calculated physicochemical and ADME/toxicity (ADMET) properties, together with thousands of molecular descriptors calculated for each compound. Biochemical kinase inhibitory assays on selected, cloned kinase enzymes for a few hundred NCL compound sets can provide sufficient biological data for rational computerized design of new analogues, based on our pharmacophore model-generating 3DNET4W QSPAR (quantitative structure-property/activity relationships) approach. Using this pharmacophore modeling approach and the ADMET filters, we can preselect synthesizable compounds for hit-and-lead optimization. Starting from this point and integrating the information from QSPAR, high-quality leads can be generated within a small number of optimization cycles. Applying NCL technology we have developed lead compounds for several validated kinase targets.

Synthesis of selective SRPK-1 inhibitors: novel tricyclic quinoxaline derivatives.

SR protein-specific kinase-1 (SRPK-1) has been identified as a validated target for hepatitis B virus (HBV). A series of novel tricyclic quinoxaline derivatives was designed and synthesised as potential kinase inhibitory antiviral agents and was found to be active and selective for SRPK-1 kinase. Most of these novel compounds have drug-like properties according to experimentally determined LogP and LogS values.