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Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

We report on the design, synthesis, and structure activity relationship studies of novel Org 27569 analogues as potential allosteric modulators of the CB1 receptors. We also investigated by computer modelling the possible location of the allosteric site on CB1 and the binding confirmation of the allosteric ligands. Docking of the synthesised molecules is also performed and the results are compared to the results of the biological bioassays. The synthesis of non-fused indole analogues of Org 27569 is described. These analogues were systematically varied to study the importance of key functional groups for CB1 allosteric activity. It was found that the two NH groups of the indole derivatives are required for activity. Activity is also significantly improved for analogues possessing a hydroxymethyl group or a hydrophobic chain at position 3 of the indole moiety. SAR analysis also shows that the presence of a dialkylamino group at the <i>para-</i>position on the aromatic side chain further improves the activity. Conformationally restricted analogues (fused indoles) of Org 27569 were prepared to determine the possible binding conformation of Org 27569.<i> </i>An analogue having the two NH groups directed in the same direction exhibited a moderate ability to enhance CP55,940 affinity and gave significant decrease in [³⁵S]GTPγS binding at 1μM, indicating the possible binding conformation for the Organon derivatives. Molecular modelling studies allowed locating a possible binding pocket for the CB1 allosteric ligands. The study described here should help the design of ligands of the CB1 allosteric site that possess higher biological activities and specificities. The results should pave the way for the discovery of the anti-obesity drugs of the future.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.

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Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xxviii, 265 p.; also includes graphics (some col.) Includes bibliographical references (p. 184-198). Available online via OhioLINK's ETD Center

Strategies for the engineering of allosteric proteins, which are proteins that bind ligands at a specific site other than the reaction site and affect the reaction activity, are still being perfected. There have been allosteric proteins successfully engineered based on the hypothesis that the two allosterically related sites are distinct, modular domains and use trial and error to construct and test novel protein domain fusions for allostery. This work uses laboratory evolution to engineer the peptide binding affinity of the protein binding domain of the allosteric E. coli protease DegS. The protein binding domain is a PDZ domain (named for Postsynaptic density protein (PSD-95), Discs-large protein (Dlg), and Zonula occludens-1 (ZO-1)) that binds the C-terminus of unfolded outer membrane porins. Combinatorial libraries of PDZ domain variants were displayed anchored to the periplasmic membrane of E. coli. The cells were permeabilized and incubated with fluorescent peptide ligands. PDZ domains were screened by flow cytometry for binding to the target peptide ligands. The PDZ domain binding affinity was improved by 20-fold for the peptide ligand that represents the physiological ligand; and the PDZ domain binding affinity was expanded to accommodate a negatively charged residue in a novel peptide ligand. The E. coli anchored peripalsmic expression (APEx) methodology in conjunction with flow cytometry had not previously been used to modify the binding affinity of a PDZ domain. The selected PDZ domain variants were then fused to the wild-type DegS protease domain and analyzed to determine if allosteric activation was made more sensitive to the native ligand or altered to respond to the novel peptide ligand. Interestingly, the DegS fusion protein with the PDZ variant containing the most subtle mutations retained a degree of allostery for the physiological peptide ligand and obtained a degree of allostery for the novel activating peptide ligand. Other selected PDZ variants with additional and expected mutations in the ligand binding site did not respond allosterically to the peptide ligands and the respective DegS fusions were constitutively active, suggesting that the amino acid network linking the allosteric binding event to protease activity is intricately integrated. / text

Today, current drug discovery and lead generation efforts focus on high throughput screening of large chemical libraries as the primary source of lead candidates. A lack of investment in novel chemotype development by pharmaceutical companies over the last 15 years coupled with the concurrent merger of screening collections and the availability of generic compound libraries commercially have resulted in many discovery efforts that lack uniqueness and do not offer a strong patent position to operate. The need for better, more diverse, and more drug-like libraries is essential in order to feed high throughput screening efforts with molecules that probe new dimensions of chemical space and allow for the discovery of untapped intellectual property.This dissertation details a complete structure based study to design novel inhibitors of B-Raf and p38a MAP Kinase. A structural evaluation of the important and similar interactions necessary for DFG-out allosteric inhibitors to bind their respective targets was accomplished through the synthesis and evaluation of three known allosteric kinase inhibitors, Gleevec®, Nexavar®, and BIRB-796, and 8 additional DFG-out allosteric inhibitors that were developed directly from fragments of these successful scaffolds. The structural insight that was gained from the evaluation of known DFG-out allosteric inhibitors was then utilized to design novel inhibitors that incorporated two unique scaffolds based on two new [3+2] cycloaddition reactions.A pyrrolo-3,4-dicarboximide scaffold has been developed through the utilization of a novel tandem [3+2] cycloaddition then elimination reaction scheme. This scaffold, which contains three sites for variation, was then rationally incorporated into lead molecules using structure-based methods and in silico feedback for the production of dual DFG-out allosteric kinase inhibitors of p38a and B-Raf kinase. These inhibitors display micromolar to submicromolar enzymatic IC50's for both p38a and B-Raf kinase and low micromolar inhibition of cell growth in 4 separate cancer cell lines.We also explored new chemistry that utilizes a key one pot, [3+2] cycloaddition reaction to obtain highly substituted imidazoles and their application in the design of specific allosteric B-Raf inhibitors. Inhibitors based on this scaffold display subnanomolar potency and a favorable kinase profile.

There has been progress towards elucidating the mechanism of Escherichia coli glycerol kinase (EcGK) control by its allosteric effectors fructose-1,6-bisphosphate (FBP) and IIAglc (a member of the phosphoenolpyruvate:glycose phosphotransferase system). Determining the mechanism requires analysis of the interaction between these effectors and the substrates of EcGK. In this study, a structural and kinetic approach was used to determine inhibition by both the effectors. For this work, the use of fluorescence anisotropy to observe ligand binding was investigated. Also, a foundation was laid for future NMR experiments with EcGK. For fluorescence studies, E36C EcGK was labeled with fluorescein and tested for changes in anisotropy in the presence of different ligands. To ensure that E36C was an appropriate representative of wildtype protein, initial velocity, inhibition, and heterotropic coupling assays were performed. Groundwork for future NMR experiments required analyzing substitutions of the native EcGK cysteines by initial velocity and inhibition studies. By comparing wildtype enzyme and E36C (variant of wildtype with an engineered cysteine residue at position 36), it was found that E36C is a suitable substitute and was not drastically affected by labeling with fluorescein. Anisotropy values differed upon binding of different ligands and enabled titrations of the enzyme substrate complexes with both effectors to obtain dissociation constants. This supports using the stopped-flow method to assess the on- and off- rates of substrates and to obtain values for Q coupling. Furthermore, the results for FBP showed that inhibition by FBP is K-type (affects affinity) with respect to ATP and V-type (affects enzyme velocity) with respect to ADP. The findings presented also showed that native cysteine substitutions effect some of the catalytic and allosteric parameters of EcGK and would be powerful reporters for ligand binding in NMR. However, the enzymes are unstable and new protocols for protein isolation will need to be drafted.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 166-178).