A Dynamic View Of ATP-Coupled Functioning Cycle Of Hsp90 And The Discovery Of Allosteric Inhibitors

Heat-shock protein 90(Hsp90)is one of the most important chaperones,which achieves its function by facilitating maturation,stabilization,activation and intracellular sorting of ~300 client proteins including hormone receptors,transcription factors,cell cycle regulatory proteins and kinases etc.Quite a few of the client proteins of Hsp90 are oncogenic,and Hsp90 plays a pivotal role in tumorigenesis due to its involvement in the regulation of cellular homeostasis of oncogenic clients.Hsp90 is serving as a promising target for anti-cancer drug development.Hsp90 consists of three major structural domains: the highly conserved N-terminal domain with ATPase activity and co-chaperone recognition function;the middle domain which carries client protein and co-chaperone recognition function;and the C-terminal domain which mediates formation of Hsp90 homodimer.The functioning cycle of Hsp90 is finely tuned by both the endogenous small molecules(ATP and ADP)and bio-macromolecules(co-chaperones including p23,Cdc37 and Aha1 etc).The ATPase activity of Hsp90 is intimately coupled to its chaperoning function,and the binding and hydrolysis of ATP will induce a series of function-related conformational changes of the N-terminal domain and also the middle domain of the chaperone.However,the dynamic molecular mechanism for the ATP-coupled functioning cycle of Hsp90 is still not fully understood.Since the ATPase activity of Hsp90 plays an important role in its functional display,the ATP-competitive inhibitors targeting the ATP-binding pocket of Hsp90 are the dominant class of Hsp90-targeted anti-cancer drug candidates.Until the year of 2015,a total number of 17 ATP-competitive inhibitors of Hsp90 have progressed into the different phases of clinical trial.However,none of them has been officially approved for cancer therapy,and the significant side effects of these inhibitors are one of the major causes.Here in this thesis,to achieve a dynamic view of the ATP-coupled functioning cycle of Hsp90 N-terminal domain,we used NMR techniques to investigate the structural characteristics and dynamic behaviors of the N-terminal domain in the absence and presence of either the non-hydrolysable AMPPCP(ATP analogue)or ADP.We then developed Hsp90 middle domain-targeted anti-cancer inhibitors by a combination use of experimental screening and structure-based medicinal chemistry optimization.And in the further studies,we tried to elucidate the working mechanisms of the active compounds and explore the structural features of the Hsp90 middle domain associated with its different functional states by using the allosteric inhibitors as probes.The novel type of anti-cancer inhibitors might progress to anti-cancer drug candidates with lesser side effects.In the first part of the thesis,to achieve a dynamic view of the ATP-coupled functioning cycle of Hsp90 N-terminal domain,we used NMR techniques to investigate the structural characteristics and dynamic behaviors of the N-terminal domain in the absence and presence of either the non-hydrolysable AMPPCP(ATP analogue)or ADP.The data from [1H,15N] HSQC titration experiments,protein dynamics analysis and NMR line-shape analysis indicate that AMPPCP and ADP bind to almost the same region of Hsp90 N-terminal domain but show different effects on the local conformation of its lid segment.The conformational exchanges in ?s-ms time scale of the mobile lid segment are promoted by the AMPPCP binding and inhibited with the addition of ADP.Consistent with the NMR data,significantly different contributions of entropy change to Gibbs free energy change were observed in the isothermal titration calorimetry experiments for Hsp90:AMPPCP and Hsp90:ADP systems,suggesting the AMPPCP binding and the ADP binding cause different effects on the local conformations of Hsp90.Using all our data and the previous findings a detailed working model for the ATP-dependent functioning cycle of Hsp90 was proposed.The N-terminal domain of apo Hsp90 adopts a cluster of inactive conformations in its resting state in solution.However,the mobile lid segment spanning A111-G135 experiencing micro-to millisecond internal motions entails the existence of transient conformations with higher energy at a low population.The ATP binding enhances the slow-motion-featured conformational exchanges of the residues spanning A117-A141 within the lid segment and region around them and promotes the sparsely-populated active conformers,although the dominant conformations of ATP-bound Hsp90 are still inactive.With the coordination of the middle domain of Hsp90 and/or cofactors,the Nterminal domain forms an active homodimer.After the accomplishment of the chaperoning tasks and the hydrolysis of ATP molecule,the dimeric N-terminal domain of Hsp90 dissociates to ADP-bound monomer state,which is favored in the inactive conformations.In the following studies,[1H,15N] HSQC titration experiments using Hsp90α NMΔ as the observation sample were carried out to unravel the allosteric modulation effects of the middle domain on the ADP/ATP(AMPPCP)recognition processes of Hsp90.The data suggests that the middle domain of Hsp90 shows no significant modulation effects on the binding of AMPPCP and ADP to Hsp90 Nterminal domain,and the perturbed residues by the binding of AMPPCP or ADP mainly focus on the ATP-binding pocket of the N-terminal domain of the chaperone.In the second part of the thesis,we carried out Hsp90 middle domain-targeted hit compound screening by using NMR techniques,and obtained one hit compound 1-E6.After that,we characterized the interactions between Hsp90 middle domain and 1-E6,and the information achieved was then applied to guide the structure-based medicinal chemistry optimization of 1-E6.Two active compounds including 16-171 and 16-175 with their binding affinities to Hsp90 middle domain at the level of hundreds of ?M were obtained.In the following studies,the molecular working mechanisms of two active compounds were elucidated by using NMR techniques.The NMR data indicate that these two compounds regulate the function display of Hsp90 by allosterically modulating the conformation of Hsp90 N-terminal domain.The induced conformation of Hsp90 N-terminal domain by the co-existence of the middle domain of the chaperone nearly diminishes with the presence of the active compound.Besides,the presence of the co-chaperone Aha1 shows no significant effects on the allosteric conformation changes of Hsp90 N-terminal domain induced by the binding of the active compound.However,when the other co-chaperone P23 is added into the system,the allosteric modulation effects caused by the addition of the active compound is totally abolished.Finally,the results of tumor cell growth inhibition assays reveal that 16-171 and 16-175 present inhibition activities at the level of tens of ?M to hundreds of ?M against human breast cancer cell line of SKBR3.