Harvard Medical School

Start : January 2015 | Status : Complete

The scientists: Dr. Hugo Fraga is a PhD Research Associate at Harvard Medical School working in Dr. Goldberg´s lab in the Cell Biology department. He has extensive experience researching the structure of ClpP, including recently in Grenoble, France. Annie Park is a graduate student in the Biological and Biomedical Sciences program at Harvard’s Graduate school of Arts and Sciences.

The sponsors: Harvard Medical School has been synonymous with excellence in education and research for generations. Dr. Alfred Goldberg has been associated with Harvard during his entire academic career and is currently professor of Cell Biology. His lab has long been a leading center for studies of the regulation and mechanisms of protein breakdown in animal and bacterial cells.

Foundation funding: The Foundation is providing £ 238,110 in support.

GSK’s contribution: GSK is providing in-kind contributions including access to the GSK´s collection of compounds, scientific expertise in HTS enzymatic campaigns and antitubercular characterization capacity of the hits identified.

Project Description: The project objective is the screening for inhibitors of the function or formation of the ClpC1P1P2 complex from M. tuberculosis (M.tb).
The blocking of ClpP1 or ClpP2 function or its regulatory ATPases, ClpX, and ClpC, rapidly kills growing and non-growing M.tb. Recently, two novel cyclic peptide antibiotics (lassomycin and ecumicin) were shown to selectively kill M.tb by activating ClpC1 ATPase, uncoupling ATP hydrolysis from protein degradation and thus preventing the regulated, breakdown of cell proteins. These findings provide strong validation for these targets and the rationale for this project.

The project team will screen for small molecules that block ATP-dependent degradation of a fluorescent protein substrate by the ClpC1P1P2 complex. This assay will screen for inhibitors of ClpP1P2 activity, its association with ClpC, as well as ClpC’s multiple functions (ATPase unfolding, protease activation).
For confirmed hits, we shall determine which component of the ClpC1P1P2 complex is affected. Possible effects on the ClpP1P2 proteolytic activity will be tested using isolated ClpP1P2 and a sensitive fluorescent substrate. Effects on ClpC will be tested by assaying ATP hydrolysis. Disruption of the interaction between the ATPase and protease would result in blockage of protein  degradation without effects on peptide or ATP hydrolysis.