A synthetic compound that potentiates bone morphogenetic protein-2-induced transdifferentiation of myoblasts into the osteoblastic phenotype

金沢大学附属病院リハビリテーション部There is an urgent need to develop methods that lower costs of using recombinant human bone morphogenetic proteins (BMPs) to promote bone induction. In this study, we demonstrate the osteogenic effect of a low-molecular weight compound, SVAK-12, that potentiated the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. Here, we report a specific compound, SVAK-12, which was selected based on in silico screenings of small-molecule databases using the homology modeled interaction motif of Smurf1-WW2 domain. The enhancement of BMP-2 activity by SVAK-12 was characterized by evaluating a BMP-specific reporter activity and by monitoring the BMP-2-induced expression of mRNA for osteocalcin and alkaline phosphatase (ALP), which are widely accepted marker genes of osteoblast differentiation. Finally, we confirmed these results by also measuring the enhancement of BMP-2-induced activity of ALP. Smurf1 is an E3 ligase that targets osteogenic Smads for ubiquitin-mediated proteasomal degradation. Smurf1 is an interesting potential target to enhance bone formation based on the positive effects on bone of proteins that block Smurf1-binding to Smad targets or in Smurf1-/- knockout mice. Since Smads bind Smurf1 via its WW2 domain, we performed in silico screening to identify compounds that might interact with the Smurf1-WW2 domain. We recently reported the activity of a compound, SVAK-3. However, SVAK-3, while exhibiting BMP-potentiating activity, was not stable and thus warranted a new search for a more stable and efficacious compound among a selected group of candidates. In addition to being more stable, SVAK-12 exhibited a dose-dependent activity in inducing osteoblastic differentiation of myoblastic C2C12 cells even when multiple markers of the osteoblastic phenotype were parallelly monitored. © 2010 Springer Science+Business Media, LLC

A Novel Selective JAK2 Inhibitor Identified Using Pharmacological Interactions

The JAK2/STAT signaling pathway mediates cytokine receptor signals that are involved in cell growth, survival and homeostasis. JAK2 is a member of the Janus kinase (JAK) family and aberrant JAK2/STAT is involved with various diseases, making the pathway a therapeutic target. The similarity between the ATP binding site of protein kinases has made development of specific inhibitors difficult. Current JAK2 inhibitors are not selective and produce unwanted side effects. It is thought that increasing selectivity of kinase inhibitors may reduce the side effects seen with current treatment options. Thus, there is a great need for a selective JAK inhibitor. In this study, we identified a JAK2 specific inhibitor. We first identified key pharmacological interactions in the JAK2 binding site by analyzing known JAK2 inhibitors. Then, we performed structure-based virtual screening and filtered compounds based on their pharmacological interactions and identified compound NSC13626 as a potential JAK2 inhibitor. Results of enzymatic assays revealed that against a panel of kinases, compound NSC13626 is a JAK2 inhibitor and has high selectivity toward the JAK2 and JAK3 isozymes. Our cellular assays revealed that compound NSC13626 inhibits colorectal cancer cell (CRC) growth by downregulating phosphorylation of STAT3 and arresting the cell cycle in the S phase. Thus, we believe that compound NSC13626 has potential to be further optimized as a selective JAK2 drug

Computational studies on protein-ligand docking

This thesis describes the development and refinement of a number of techniques for molecular docking and ligand database screening, as well as the application of these techniques to predict the structures of several protein-ligand complexes and to discover novel ligands of an important receptor protein. Global energy optimisation by Monte-Carlo minimisation in internal co-ordinates was used to predict bound conformations of eight protein-ligand complexes. Experimental X-ray crystallography structures became available after the predictions were made. Comparison with the X-ray structures showed that the docking procedure placed 30 to 70% of the ligand molecule correctly within 1.5A from the native structure. The discrimination potential for identification of high-affinity ligands was derived and optimised using a large set of available protein-ligand complex structures. A fast boundary-element solvation electrostatic calculation algorithm was implemented to evaluate the solvation component of the discrimination potential. An accelerated docking procedure utilising pre-calculated grid potentials was developed and tested. For 23 receptors and 63 ligands extracted from X-ray structures, the docking and discrimination protocol was capable of correct identification of the majority of native receptor-ligand couples. 51 complexes with known structures were predicted. 35 predictions were within 3A from the native structure, giving correct overall positioning of the ligand, and 26 were within 2A, reproducing a detailed picture of the receptor-ligand interaction. Docking and ligand discrimination potential evaluation was applied to screen the database of more than 150000 commercially available compounds for binding to the fibroblast growth factor receptor tyrosine kinase, the protein implicated in several pathological cell growth aberrations. As expected, a number of compounds selected by the screening protocol turned out to be known inhibitors of the tyrosine kinases. 49 putative novel ligands identified by the screening protocol were experimentally tested and five compounds have shown inhibition of phosphorylation activity of the kinase. These compounds can be used as leads for further drug development

Development and Characterization of Tool Compounds Targeting the Runt Domain’s interaction With Cbfβ

RUNX1 and CBFβ, which encode subunits of the core binding factor, are frequent targets of chromosomal aberrations in hematological malignancies. We previously determined that CBFβ (encoded by CBFB) is important for the transforming activity of the chimeric protein AML1-ETO protein (RUNX1-RUNX1T1) generated by the t(8;21), and other studies showed that normal Runx1 functions are essential for survival and maintenance of some leukemias lacking RUNX1 or CBFB mutations. Thus, we hypothesized that we could achieve therapeutic efficacy in multiple leukemias by targeting the Runx1:CBFβinteraction with small molecules. Using the structural information of the DNA binding Runt domain (RD) of Runx1 and its interface with CBFβ, we employed a computational screen for a library of 78,000 drug-like compounds, and further optimized our leads. The Runt domain inhibitors (RDIs) bind directly to the RD and disrupt its interaction with CBFβ. We showed that the RDIs reduced growth and induced apoptosis of t(8;21) acute myeloid leukemia (AML) cell lines, and reduced the progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. The RDIs had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines that did not harbor the t(8;21). Furthermore, our inclusion of a structurally related and weakly active compound as a control strongly support that the efficacies we observed were due to on target inhibition of RUNX functions. Our results confirmed that the RDIs might prove efficacious in various AMLs, and that a therapeutic window is available to specifically target malignant cells. We developed a pro-drug AI-9-59 with improved solubility and pharmacokinetic properties and assessed whether it has any in vivo efficacies in mouse leukemia models. Our results showed that the pro-drug was toxic to mice at dosage above 50 mg/kg and had no observable growth inhibitory effect on leukemia cells, suggesting that the concentration of the pro-drug necessary to inhibit endogenous core binding factor activity exceeds the maximum tolerated dose in mice. However, the expansion of granulocyte macrophage progenitors, and the gastrointestinal toxicity phenotype we observed suggested that the effects could be from on-target repression of RUNX proteins functions

COMBINATORIAL LIBRARY DESIGN OF MUTATION-RESISTANT HIV PROTEASE INHIBITORS.

The emergence of HIV strains that are resistant to current HIV protease inhibitors in the past few years has become a major concern in AIDS treatment. The goal of this project is to design a combinatorial library of potential lead compounds that can bind to both the wild-type and mutant proteases and that can resist further mutations. A recent crystallographic study of complexes of HIV protease with its substrates has provided structural insights into the differential recognition of the substrates and inhibitors. It has been proposed that clinical resistance is a consequence of inhibitors failure to stay within the consensus substrate volume. In this work, we devised a quantitative indicator of the degree to which a candidate ligand falls outside the consensus substrate volume, and determined its correlation with the inhibitor's sensitivity to clinically relevant resistant mutations. The validation of this hypothesis has encouraged us to use this strategy in our design of a combinatorial library of inhibitors. The compounds in a typical combinatorial library are built around a common structural scaffold possessing multiple connection points where substituents can be added by reliable synthetic steps. As the number of compounds encompassed by such a combinatorial scheme frequently exceeds what can actually be synthesized and tested, virtual screening methods are sought to shortlist the compounds. Even though these methods require only seconds to minutes of CPU time per compound, exhaustive screening of an entire virtual combinatorial library is computationally demanding. We therefore implemented a simple algorithm of combining substituents that have been optimized independently for the substituent sites. This method was compared with Genetic Algorithm, a global optimization method and was found equally efficient. This simple method was hence chosen for the design process. A combinatorial library based on these ideas and methods has been synthesized and tested. It includes four compounds with nanomolar inhibition constants. Two of them were shown to have retained affinity against a panel of treatment-resistant mutations

Lead optimization for new antimalarials and Successful lead identification for metalloproteinases: A Fragment-based approach Using Virtual Screening

Lead optimization for new antimalarials and Successful lead identification for metalloproteinases: A Fragment-based approach Using Virtual Screening Computer-aided drug design is an essential part of the modern medicinal chemistry, and has led to the acceleration of many projects. The herein described thesis presents examples for its application in the field of lead optimization and lead identification for three metalloproteins. DOXP-reductoisomerase (DXR) is a key enzyme of the mevalonate independent isoprenoid biosynthesis. Structure-activity relationships for 43 DXR inhibitors are established, derived from protein-based docking, ligand-based 3D QSAR and a combination of both approaches as realized by AFMoC. As part of an effort to optimize the properties of the established inhibitor Fosmidomycin, analogues have been synthesized and tested to gain further insights into the primary determinants of structural affinity. Unfortunately, these structures still leave the active Fosmidomycin conformation and detailed reaction mechanism undetermined. This fact, together with the small inhibitor data set provides a major challenge for presently available docking programs and 3D QSAR tools. Using the recently developed protein tailored scoring protocol AFMoC precise prediction of binding affinities for related ligands as well as the capability to estimate the affinities of structurally distinct inhibitors has been achieved. Farnesyltransferase is a zinc-metallo enzyme that catalyzes the posttranslational modification of numerous proteins involved in intracellular signal transduction. The development of farnesyltransferase inhibitors is directed towards the so-called non-thiol inhibitors because of adverse drug effects connected to free thiols. A first step on the way to non-thiol farnesyltransferase inhibitors was the development of an CAAX-benzophenone peptidomimetic based on a pharmacophore model. On its basis bisubstrate analogues were developed as one class of non-thiol farnesyltransferase inhibitors. In further studies two aryl binding and two distinct specificity sites were postulated. Flexible docking of model compounds was applied to investigate the sub-pockets and design highly active non-thiol farnesyltransferase inhibitor. In addition to affinity, special attention was paid towards in vivo activity and species specificity. The second part of this thesis describes a possible strategy for computer-aided lead discovery. Assembling a complex ligand from simple fragments has recently been introduced as an alternative to traditional HTS. While frequently applied experimentally, only a few examples are known for computational fragment-based approaches. Mostly, computational tools are applied to compile the libraries and to finally assess the assembled ligands. Using the metalloproteinase thermolysin (TLN) as a model target, a computational fragment-based screening protocol has been established. Starting with a data set of commercially available chemical compounds, a fragment library has been compiled considering (1) fragment likeness and (2) similarity to known drugs. The library is screened for target specificity, resulting in 112 fragments to target the zinc binding area and 75 fragments targeting the hydrophobic specificity pocket of the enzyme. After analyzing the performance of multiple docking programs and scoring functions forand the most 14 candidates are selected for further analysis. Soaking experiments were performed for reference fragment to derive a general applicable crystallization protocol for TLN and subsequently for new protein-fragment complex structures. 3-Methylsaspirin could be determined to bind to TLN. Additional studies addressed a retrospective performance analysis of the applied scoring functions and modification on the screening hit. Curios about the differences of aspirin and 3-methylaspirin, 3-chloroaspirin has been synthesized and affinities could be determined to be 2.42 mM; 1.73 mM und 522 μM respectively. The results of the thesis show, that computer aided drug design approaches could successfully support projects in lead optimization and lead identification. fragments in general, the fragments derived from the screening are docke

Development of an affinity sensor for the detection of aflatoxin M1 in milk

Much research has been done on aflatoxins since their discovery in the 1960’s where it was concluded that aflatoxins have carcinogenic, mutagenic, teratogenic and immunosuppressive properties. Aflatoxin M1 exists in milk and since milk is a major component of the diet of infants, the maximum permissible limit set by the EU is 50 parts per trillion (ng L -1 ). Current methods of analysis for aflatoxin M1 is primarily based around techniques such as HPLC and TLC which require extensively trained operators and equipped laboratories. Using antibodies as receptors in an enzyme linked immunosorbent assay (ELISA), the analysis costs can be reduced and simplified, however, an equipped laboratory is still required. Hence there is a need for a low cost, rapid, portable instrument which is easy to use at the point of source for the detection of aflatoxin M1. This thesis describes the development of affinity sensors to meet these requirements. Firstly the design and optimisation of an ELISA method was carried out, utilising a commercially sourced monoclonal antibody. Once the antibodies suitability for sensing aflatoxin M1 was determined the antibody was successfully employed as the receptor for a screen printed HRP/TMB based immunosensor. Upon the analysis of milk it was observed that milk caused extensive interference and through a series of chemical extractions the interference was attributed to whey proteins in the milk with suspicion towards a- lactalbumin. A simple pre-treatment technique of adding calcium chloride was performed and the interference from the whey proteins was removed. The resulting immunosensor achieved a sensitivity of 39 ng L -1 (Figure 3.26), however, poor reproducibility was observed due to the screen printed electrode production (%CV = 21% variance for screen printed electrode production). Gold cell on a chip microelectrode arrays were used to replace the screen printed electrodes and the successful covalent attachment of the antibody to the microelectrode array through PDITC cross linking compound was monitored using atomic force microscopy and scanning electron microscopy. It was shown that the majority of the antibodies during immobilisation orientate in a ‘side on’ orientation and therefore a cheap capture polyclonal antibody was first immobilised before the addition of the sensing anti-aflatoxin M1 monoclonal antibody. Using the microelectrode array an improvement of the sensitivity as well as a reduction of the milk interference was shown. Sensitivity was improved to 8 ng L -1 in milk (Figure 4.23). Further work was performed to substitute the fragile antibody used in the sensing layer for a robust synthetic peptide receptor. Initially a virtual library of synthetic peptides was created using de novo design techniques in silico. Further computational techniques were performed to determine the best peptide from the library. This peptide had a sequence of PVGPRP. From literature a peptide (LLAR) was reported with affinity for aflatoxin B1. This sequence along with the de novo design peptide was synthesised and tested using a host of techniques and immobilisation chemistries such as optical waveguide lightmode spectroscopy (OWLS), BIAcore and enzymatic techniques using EDC/NHS, glutaraldehyde and BS 3 cross linking methods. The affinity of both peptides to aflatoxin M1 was demonstrated however further work is required to quantify the affinity and to incorporate the peptides into the microelectrode array.EThOS - Electronic Theses Online ServiceGBUnited Kingdo