Liza Ngo has completed her master’s degree from the Georgia State University. She is currently a fourth year Phd. student in the Pharmaceutical Biomedical Sciemce department at the Univeristy of Georgia. She is also awardee of the National Science Foundation Graduate Fellowship
Histone acetyltransferases (HATs) mediate the transfer of an acetyl group from the cofactor, acetyl- CoA, to the ɛ- amino group of specific lysines in diverse protein substrates, most notably nuclear histones. The deregulation of these enzymes are connected to a number of disease, such as prostate and breast cancer; however to date, there are no FDA approved drug targeting HATs. Reliable and rapid biochemical assays for HATs are critical for understanding biological functions of protein acetylation, as well as for screening small-molecule inhibitors and activators of HAT enzymes. We present the design of a scintillation proximity assay (SPA) which was used in an inhibitor library screening. The acetyl donor was [3H]Ac-CoA and a biotin-modified histone peptide served as the HAT substrate. Following the HAT reaction, guanidinium chloride was added to quench the reaction. Lastly, streptavidin-coated beads were added to induce proximity of acetylated substrate to the scintillant molecules. Overall, this microplate formatted, mix-and-measure assay showed consistent and robust performance for HAT activity measurement. The SPA was used in a p300 inhibitor screening with compounds identified from a computational screening. A few hits were identified from the p300 inhibitor screening with IC50 values in the submicromolar range. Surprisingly, when the best p300 inhibitor hits were counter screened with other HAT family members, a GCN5 activator was identified. This novel activator compound increased GCN5 activity by four-fold.
Lionel Zapata it’s a PhD strudent of the University of Concepcion. Obtained his master's degree in physiology in 2015. He is member of biotechnological company dedicated to the development of biotechnological solutions for the pharmaceutical area.
Many different monoclonal antibodies (Mabs) are already available for cancer, chronic infections and autoimmune diseases therapy. Over the last few years biopharmaceutical companies have experienced a huge improvement in the Mabs manufacturing process, however the antibody based therapies still remain too expensive. Because of the molecular nature of mAbs, the recombinant expression in mammalian cells can´t be replaced by less expensive alternatives such as bacteria or yeast culture. Here, we propose a molecular alternative for therapeutic mAbs, based on fragments of single chain antibodies (scFv) coupled through trimerization domain of tetranectine. Trimeric scFvs share with Mabs the domains involve in target recognition and have a similar molecular weigh. These trimeric scFv can be successful expressed in cheaper platforms as Yeasts or Bacteria culture. To obtain scFvs coupled trough trimerization domains the yeast expression vector pPSHG20-tetra was constructed by restriction cloning. This vector codes for the expression of an anti-VEGF scFv coupled by the tetranectine trimerization domain following by a poly histidine tag was added to N-terminal, to facilitate identification and subsequent purification. MP-36 yeast strain was transformed with the plasmid-tetra pPSHG20 by electroporation and transformed cells were isolated by auxotrophy selection and the and the insertion was checked by Southern blotting. Trimeric scFv expression was performed with 0.7% methanol during 4 days. Then trimeric scFvs were purified using affinity purification by IMAC. The protein expression yield and the ability to trimerize were analyzed by SDS-PAGE, Western blotting and densitometry, in reducing and non-reducing conditions. Finally, the bioactivity of trimeric scFv was confirmed by in vitro and in-vivo models. As a result, we demonstrated that the expresion of high yields of trimeric scFv fragments in yeast is possible. The estimated molecular weight of trimeric scFv was similar to the IgG antibody isotype. Furthermore, trimers of scFv fragments were obtained with high purity (95%) and remained their trimeric structure. The trimeric ati-VEGF scFv antibodys inhibits the biologic activity of human VEGF in in-vitro and in-vivo assay. This results suggests that functional scFv coupled trough trimerization domains production is possible. The trimeric scFv could be al reliable alternative to the Mabs based therapy with similar bioactivity but with a lower production cost.