| Popis: |
A large subset of infant acute leukaemias is associated with chromosomal translocations involving the Mixed Lineage Leukaemia (MLL) gene, which spans the breakpoint region at chromosome llq23, and is correlated with poor patient prognosis and early relapse. The marked heterogeneity, both at the clinical and molecular level, within this class of leukaemia makes defining a suitable therapy a very challenging task. A trimeric interaction of the MENIN and LEDGF proteins with the N-terminal region of MLL is responsible for the constitutive expression of downstream transcription factors (e.g. HOXA9), and is required for malignant transformation. In addition, the discovery of another protein complex formed at the C-terminal region of some of the most common MLL-fusion partners (i.e. ENL, AF9 and AF4) has revealed another possible therapeutic target. Dual-targeting strategies involving both the N- and C- terminal regions of the most common MLL-fusions might represent an exciting therapeutic opportunity. The aim of this project was to explore methods that could lead to the development of small molecule inhibitors of both MLL protein complexes and that are capable of targeting multiple different MLL malignancies regardless of the specific chromosomal translocation encountered. I developed a mammalian two- and three- hybrid system to recapitulate the MLLIMENINILEDGF and AF9/AF4IENL interactions in vivo and to map the MLL binding site on the MENIN protein. Pre-clinical inhibitors were identified using this and other cell-based platforms and through the identification of small antibody fragments and chemical compounds with potential to disrupt protein interactions. To develop reagents capable of interfering with protein complexes at the N- and C-terminal regions of MLL fusions, intracellular antibody capture (lAC) technology was used to isolate intracellular antibodies (iDabs) targeting the Integrase Binding Domain (IBD) of LEDGF or targeting AF9. To simplify and reduce the timescale of the intracellularly antibody screening process for members of protein complexes, I developed a dual screening vector to simultaneously screen two antigens of interest (in this case, parts of the MLL complex, namely IBD of LEDGF and AF9). A selection of small DNA-encoded chemical compounds has also been identified from a DNA-based diverse chemical library screen, leading to the identification of four compounds that showed sustained binding to IBD in an SPR assay. An in silica structural analysis of such compounds has predicted good binding affinity to the IBD highlighting their role as candidates for the targeting of the MLL/MENIN/LEDGF complex. In addition, to facilitate the future co- crystallisation of preclinical reagents with LEDGF for drug development strategies, I crystallised the IBD in its monomeric form, and solved the structure to 2.05 A resolution. |
