RESUMO
The role of dynamics in enzymatic function is a highly debated topic. Dihydrofolate reductase (DHFR), due to its universality and the depth with which it has been studied, is a model system in this debate. Myriad previous works have identified networks of residues in positions near to and remote from the active site that are involved in dynamics and others that are important for catalysis. For example, specific mutations on the Met20 loop in E. coli DHFR (N23PP/S148A) are known to disrupt millisecond-timescale motions and reduce catalytic activity. However, how and if networks of dynamically coupled residues influence the evolution of DHFR is still an unanswered question. In this study, we first identify, by statistical coupling analysis and molecular dynamic simulations, a network of coevolving residues, which possess increased correlated motions. We then go on to show that allosteric communication in this network is selectively knocked down in N23PP/S148A mutant E. coli DHFR. Finally, we identify two sites in the human DHFR sector which may accommodate the Met20 loop double proline mutation while preserving dynamics. These findings strongly implicate protein dynamics as a driving force for evolution.
RESUMO
A central challenge in chemical biology is to distinguish molecular families in which small structural changes trigger large changes in cell biology. Such families might be ideal scaffolds for developing cell-selective chemical effectors - for example, molecules that activate DNA damage responses in malignant cells while sparing healthy cells. Across closely related structural variants, subtle structural changes have the potential to result in contrasting bioactivity patterns across different cell types. Here, we tested a 600-compound Diversity Set of screening molecules from the Boston University Center for Molecular Discovery (BU-CMD) in a novel phospho-flow assay that tracked fundamental cell biological processes, including DNA damage response, apoptosis, M-phase cell cycle, and protein synthesis in MV411 leukemia cells. Among the chemotypes screened, synthetic congeners of the rocaglate family were especially bioactive. In follow-up studies, 37 rocaglates were selected and deeply characterized using 12 million additional cellular measurements across MV411 leukemia cells and healthy peripheral blood mononuclear cells. Of the selected rocaglates, 92% displayed significant bioactivity in human cells, and 65% selectively induced DNA damage responses in leukemia and not healthy human blood cells. Furthermore, the signaling and cell-type selectivity were connected to structural features of rocaglate subfamilies. In particular, three rocaglates from the rocaglate pyrimidinone (RP) structural subclass were the only molecules that activated exceptional DNA damage responses in leukemia cells without activating a detectable DNA damage response in healthy cells. These results indicate that the RP subset should be extensively characterized for anticancer therapeutic potential as it relates to the DNA damage response. This single cell profiling approach advances a chemical biology platform to dissect how systematic variations in chemical structure can profoundly and differentially impact basic functions of healthy and diseased cells.
