No description available.

A preorganised amino acid derivative containing a cyclopropyl constraint was designed to orient an amino acid into its bound conformation. This constrained mimic was determined by ITC to be equally potent to the native Phe derivative. It was found that a more favorable enthalpy of binding was compensated by an equally unfavorable entropy compared to the native ligand. In order to properly ascertain the effects of the cyclopropane constraint, a flexible control containing the same number of heavy atoms was synthesized and tested, and it was found to be at least 200 fold less potent than the constrained analog. However, without structural data of the flexible control, it is difficult to infer if the differences in ligand binding affinity arose from the ligand constraint or some other unknown complexity to binding. We studied the thermodynamic and structural effects of modifying alkyl chains of n-alka(e)nol and phenylalka(e)nol binders to MUP-I by both the removal of a rotor via deletion of a methylene unit and restriction of a rotor via the installation of an internal olefin. In general, we observed that a similar thermodynamic signature accompanies modifications for both the n-alka(e)nol and phenylalka(e)nol ligands: A favorable T[delta][delta]Sºo̳b̳s̳ is compensated by an unfavorable T[delta][delta]Hºo̳b̳s̳ such that T[delta][delta]Gºo̳b̳s̳ for both removal of a methylene and insertion of an internal olefin are unfavorable and equipotent, respectively. The insertion of an internal olefin into an alkyl chain led to significantly more favorable entropies than does methylene removal, yet enthalpy-entropy compensation leads to nearly equipotent binding energetics. However, we did find a strong correlation between [delta]Ho̳b̳s̳° and buried apolar Connolly Surface Area (CSA). The intrinsic free energies of introducing an internal olefin into the n-alkanols and phenylalkanols differ markedly from the observed data. It was observed that intrinsic affinities are more favorable than the observable because a favorable T[delta][delta]S⁰i̳n̳t̳ dominates an unfavorable [delta][delta]Hºi̳n̳t̳. Also, we discovered that the intrinsic entropies of inserting an internal olefin are nearly double that of removing a methylene group, suggesting that the insertion of an internal olefin results in the restriction of more C-C rotors. We have shown through ITC analysis that the added substituents probed in this study provided binding increases to our Grb2 SH2 ligands as expected, but that the thermodynamic driving force of binding affinities depended greatly upon the specific nature and flexible mobility of the ligands in the binding pocket. Through a combination of X-ray and ITC studies it was shown that ligands containing rigid and aromatic functional groups bound with a higher [delta]H° than the more flexible alkyl ligands, and that this effect correlates well with more direct vdW contacts made in the pocket. Finally, we described a case study where a strict adherence to the Topliss operational schemes led to an expedient development of novel MUP-I binding analogs. The validity of the schemes was also depicted through the synthesis and testing of ligands that were correctly predicted to be weaker/equipotent to the starting ligand. Of important note is that the degree to which the schemes led to affinity boost depended greatly on the starting potency of the initial compound. / text

Mutation and over-expression of Receptor Tyrosine Kinases (RTKs) or the proteins they regulate serve as oncogenic drivers in diverse cancers. RTKs catalyze the transfer of phosphate from ATP to the hydroxyl group on tyrosine. The proximal stretch of amino acids including this post translational modification is then able to be recognized by SH2 and PTB domains. Chapter 1 details our work to better understand RTK signaling and its link to oncogenesis using protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 out of the 53 Receptor Tyrosine Kinases. Chapter 2 expands upon this work to study the next layer of binding among SH2 and PTB domain-containing adaptor proteins themselves. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins driving oncogenesis, including both receptors and adaptor proteins, tend to be highly interconnected via a network of SH2 and PTB domain-mediated interactions. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in these well-studied signaling networks. Despite the extensive work presented here on experimentally determining interactions, we nevertheless are unable to keep up with the discovery of new sites of tyrosine phosphorylation by high throughput mass spectrometry as well as their mutation in cancer discovered by next generation tumor sequencing approaches. Chapter 3 introduces work in progress to build a unified predictive model of SH2 domain interactions via integration of diverse data sets of binding as well as crystal structures of domain-peptide interactions. This model will enable researchers discovering new phosphorylation events or mutations to be able to predict potential interaction partners and thereby elucidate novel functional mechanisms. / Chemistry and Chemical Biology

No description available.

No description available.

A series of phosphotripeptide ligands for the Grb2 SH2 domain was designed and synthesized, each of which derived from the minimal consensus sequence required for binding: Ac-pYXN. The binding affinity and related thermodynamic parameters were determined by isothermal titration calorimetry. Both the size and connectivity of the side-chain was varied. The consequences of incorporating α,α-disubstitution at the pY+1 residue on binding thermodynamics were evaluated, as were the effects of constraining the side-chains in a ring. The series was evaluated from a number of perspectives: (1) increasing size of the pY+1 residue by utilizing various amino acid types: monoalkyl, dialkyl, or cycloalkyl; (2) comparisons between ligands with the same number of carbons (scission control); and (3) by comparing ligands incorporating cyclic pY+1 residues with those incorporating α,α-dialkyl residues with one fewer methylene group (excision control). Inconsistencies in the thermodynamic consequence of constraining the backbone were observed within this set of ligands, which reveal the limitations of our understanding of protein-ligand interactions. Aspects of both the classical and non-classical hydrophobic effect were observed, but the occurance of one over the other could not be explained. / text

The cardiovascular system develops through two distinct processes in embryogenesis: vasculogenesis, whereby the primary plexus in the heart is formed along with embryonic and extraembryonic vasculature, and angiogenesis, which begins after vasculogenesis and results in the refinement and maturation of the branched vessel system. In pathological angiogenesis, tumors expand by releasing pro-angiogenic factors in response to hypoxic conditions. The Tie receptors, Tie1 and Tie2, are receptor tyrosine kinases that are integral to angiogenic pathways. A family of Angiopoietins, Ang1-4, have been shown to act as ligands for Tie2, of which Ang1 and Ang2 are best characterized. Activation of the receptor causes dimerization and autophosphorylation, whereby adaptor proteins recognizing the phosphorylated tyrosine activate downstream signaling via their Src homology 2 (SH2) domains. Currently there are no phosphospecific antibodies for Tie2, therefore, identifying critical residues responsible for certain pathways remains difficult. In our study, we aim to use purified SH2 domains of known binding partners to Tie2 to assess the phosphorylation state of the receptor under various cellular conditions and settings, utilizing immunoprecipitation and western blotting. Unexpectedly, we found that Tie2 can bind non-specifically to nickel sepharose when the SH2 proteins were used as antibody mimetics, and was unable to be consistently precipitated in Protein A sepharose when used in conjunction with a monoclonal YFP antibody. Under the latter conditions however we were able to precipitate the SH2 protein itself. When immunoprecipitations were used with cobalt activated IMAC beads, we were able to precipitate Tie2 in overexpressed systems using the SH2 domains of Shp2 N-C and Grb2. As expected, phosphorylation of Tie2 in the presence of its orphan receptor Tie1 was attenuated compared to wild-type levels. Based upon available data, we anticipate this method as a useful tool to assess the phosphorylation state of Tie2 and its signaling pathways in the near future.

SRMS (Src-Related tyrosine kinases lacking C-terminal Regulatory tyrosine and N terminal Myristoylation Sites) belongs to a family of non-receptor tyrosine kinases, which also includes breast tumor kinase (BRK). SRMS was first identified in 1994 in a screen for the genes that regulate the growth and differentiation of neuroepithelial cells. This 54 kDa protein spanning 488 amino acids, consists of the prototypical Src homology 3 (SH3), Src homology 2 (SH2) and a tyrosine kinase domain. While BRK has been documented for its expression in over 60 % of breast carcinomas, information on SRMS on similar grounds remains absent from the literature. Furthermore, unlike BRK, knowledge of how SRMS regulates its enzymatic activity as well as the identification of its substrates remains unknown. The work in this thesis demonstrates that SRMS is potentially expressed in the majority of breast carcinomas. To understand the biochemical and cellular functions of SRMS, a series of mutants comprising point mutations as well as the deletion of the N-terminal region and the functional, SH3 and SH2 domains, were generated and assessed for enzymatic activity in cells. This study demonstrates for the first time that the wild type protein is apparently constitutively active and that its N-terminal region regulates its enzymatic activity. As well, three critical amino acid residues in the protein namely, lysine 258 (ATP binding site), tyrosine 380 (auto-phosphorylation site) and tryptophan 223 (intramolecular interaction) have been characterized. All three residues have been determined to be essential for the enzymatic activity of SRMS. Finally, the adapter protein Dok1 has been characterized as a novel substrate of SRMS. The results from the present study underscore the potential significance of the catalytically active non-receptor tyrosine kinase, SRMS that should serve as a foundation upon which further research may ensue in the context of breast tumorigenesis.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.