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11	Exploring Key Orientations of Small Molecules to Disrupt Protein-protein Interactions Ko, Eunhwa 2012 May 1900 (has links) Protein-protein interactions (PPIs) are attractive targets because of their therapeutic potential. One approach to design small molecules that can disrupt the PPIs is to use structural information of proteins. With this approach, triazole-based peptidomimetics that mimic beta-turn hot-spot regions in neurotrophins were synthesized. The monovalent mimics were assembled into bivalent mimics via a combinatorial method. Three different bivalent mimics were prepared for different studies. Bivalent mimics with long-linkers bound to TrkA or TrkC receptor and showed partial antagonism for the receptors. Other mimics were conjugated with cytotoxic compounds and they were used for TrkC targeted drug delivery. The last group of bivalent mimics previously showed targeted delivery effects for pancreatic cancer cells. In this study, we synthesized Eu-chelated bivalent mimics to perform a competitive binding assay for pancreatic cancer cells. Previous research in our group focused on design of secondary structures' mimics on rigid scaffolds as "minimalist mimics." We sought to establish structural design criteria for the minimalist mimics, and we wanted to propose that sets of such compounds could mimic local pairs of amino acids in any secondary structures as "universal peptidomimetics." Thus, we designed five compounds, such as oxazoline-, pyrrole-, dyine- "kinked" and "linear" bistrizole-based peptidomimetics, and performed molecular modelings, DFT calculations, and QMD for them to validate our hypothesis. On the concepts of "minimalist mimics" and "universal peptidomimetics," we developed the C alpha ? C beta vector matching program to evaluate preferred orientations of C alpha - C beta coordinates for secondary structures. We applied the program to omegatides and pyrrolinone-pyrrolidine oligomers. The compounds matched better with strands than for helices. We expanded the C alpha ? C beta vector matching idea to a method that ranks preferred conformations of small molecules on any combination of three interface side-chains in all structurally characterized PPIs. We developed a PDB mining program (explores key orientation, EKO) to do this, and EKO applied to pyrrolinone-pyrrolidine oligomers to find targets. EKO found several interesting targets, such as AICAR Tfase, GAPDH, and HIV-1 protease. HIV-1 dimerization inhibition and Zhang-Poorman kinetic assays were performed to validate our hypothesis, and the results showed that pyrrolinone-pyrrolidine derivatives inhibited HIV-1 dimerization. Explores Key Orientation Universal Peptidomimetics minimalist mimics protein-protein interactions TrkC targeted drug delivery triazole-based beta-turn mimics bivalent mimics
12	Micro RNA-Mediated regulation of the full-length and truncated isoforms of human neurotrophic tyrosine kinase receptor type 3 (NTRK 3) Guidi, Mònica 13 January 2009 (has links) Neurotrophins and their receptors are key molecules in the development of thenervous system. Neurotrophin-3 binds preferentially to its high-affinity receptorNTRK3, which exists in two major isoforms in humans, the full-length kinaseactiveform (150 kDa) and a truncated non-catalytic form (50 kDa). The twovariants show different 3'UTR regions, indicating that they might be differentiallyregulated at the post-transcriptional level. In this work we explore howmicroRNAs take part in the regulation of full-length and truncated NTRK3,demonstrating that the two isoforms are targeted by different sets of microRNAs.We analyze the physiological consequences of the overexpression of some of theregulating microRNAs in human neuroblastoma cells. Finally, we providepreliminary evidence for a possible involvement of miR-124 - a microRNA with noputative target site in either NTRK3 isoform - in the control of the alternativespicing of NTRK3 through the downregulation of the splicing repressor PTBP1. / Las neurotrofinas y sus receptores constituyen una familia de factores crucialespara el desarrollo del sistema nervioso. La neurotrofina 3 ejerce su funciónprincipalmente a través de una unión de gran afinidad al receptor NTRK3, del cualse conocen dos isoformas principales, una larga de 150KDa con actividad de tipotirosina kinasa y una truncada de 50KDa sin dicha actividad. Estas dos isoformasno comparten la misma región 3'UTR, lo que sugiere la existencia de unaregulación postranscripcional diferente. En el presente trabajo se ha exploradocomo los microRNAs intervienen en la regulación de NTRK3, demostrando que lasdos isoformas son reguladas por diferentes miRNAs. Se han analizado lasconsecuencias fisiológicas de la sobrexpresión de dichos microRNAs utilizandocélulas de neuroblastoma. Finalmente, se ha estudiado la posible implicación delmicroRNA miR-124 en el control del splicing alternativo de NTRK3 a través de laregulación de represor de splicing PTBP1. RISC complex retinoic acid renilla luciferase real-time quantitative PCR PTBP1 pri-miRNA pre-miRNA post-transcriptional regulation piRNA PicTar pGL4.13 PCR P-body p75NTR overexpression NTRK3 NTRK2 NTRK1 non-coding RNAs NGF neurotrophin neurotrophic signaling neuronal differentiation neurodevelopment neuroblastoma nervous system mRNA miRNA target prediction miRNA profiling miRNA mimic miRNA microarray miRNA inhibitor miRanda microRNA microarray luciferase assay Ingenuity Pathway Analysis (IPA) heLa cells gene silencing gene regulation full-length isoform (FL-NTRK3) firefly luciferase ERK disease dicer cancer development BDNF argonaute alternative splicing 3'UTR RNAhybrid RT-PCR SH-SY5Y cells siRNA standard error synaptic plasticity target validation TargetScan transfection translational repression TrkA TrkB TrkC truncated isoform (TR-NTRK3) tyrosine kinase (TK) western blot 575 61

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Exploring Key Orientations of Small Molecules to Disrupt Protein-protein Interactions

Micro RNA-Mediated regulation of the full-length and truncated isoforms of human neurotrophic tyrosine kinase receptor type 3 (NTRK 3)