User:MSUgraduatestudents/Hsp90 inhibitors

Hsp 90 as an Antitumor Drug Target

Hsp90 keeps the death protiens in an apoptosis resistant state by direct assosciation. This is achieved by being a chaperone protein to several client proteins that play a central pathogenic role in human diseases, including cancer, neurodegenerative diseases and viral infections. Geldanamycin has the capability to directly bind to the ATP-binding pocket at the N-terminal domain of Hsp90 and inhibit the release of the receptor Hsp90. Hsp90 is also a promising therapeutic approach for Ewing's Sarcoma (ES) since it shows several deregulated autocrine loops mediating cell survival and proliferation. Inhibition of Hsp90 causes proteosomal destruction of client proteins Akt, KIT, and IGF1R, which could be due to precluding physical contact between client proteins and Hsp90. So since the molecular chaperones are overexpressed in a wide variety of cancer cells and in virally transformed cells, inhibiting the function of these chaperones is essential to controlling cancer cells, as this would affect the activity of signaling proteins. The availability of drugs that can specifically target Hsp90 and inhibit its function, resulting in the depletion of client proteins, has made Hsp90 a novel and exciting target for cancer therapy.

Natural Product Inhibitors

The current Hsp90 inhibitors are developed from Geldanamycin (GM) and Radicicol (RD), which are the natural product inhibitors and are starting point for new approach. Hsp90 is required for ATP dependent refolding of denatured or unfolded proteins and for the conformational maturation of a subset of proteins involved in the response of cells to extracellular signals. Steroid receptors Raf-1, Atk, Met, and Her 2 have a conserved unique pocket in teh N-terminal region that binds ATP, ADP and weak ATPase activity. GM and RD tightly bind to this pocket and prevent the release of protein from chaperone complex. Thus the protein cannot achieve native conformation and is degraded by proteosome. Addition of such inhibitor causes proteosomal degradation of signaling proteins like steroid receptors, Raf kinase and Akt. GM and RD also inhibit mutated protein in cancer cells like P53, Vsrc, BCR–ABL. GM is an effective Hsp90 inhibitor still it cannot be used in vivo because of its high toxicity and liver damage ability.

Geldanamycin derivative 17 AAG 

17AAG is the semisynthetic derivative of natural product Geldanamycin. It is less toxic with same therapeutic potential as GM. It is the first HSP90 inhibitor to be evaluated in clinical trials. Currently 17AAG is being evaluated as potent drug against Acute Myelogenous Leukemia (AML). 17 AAG decreases the concentration of client proteins but it was a question of debate if 17 AAG affected the genes for client proteins or it inhibited cytosolic proteins.

Purine Scaffolding

One of the important results obtained from the study of natural product inhibitor Geldanamycin and its interaction with HSP90 is that the use of smaller molecules as inhibitors instead of complex molecules like radicicol is more efficient. Based on this information and advanced rational drug design technique, pharmacologically relevant scaffolds can be constructed. PU3 has a structural resemblance with ATP which is natural ligand for N terminal domain.X-ray crystallography data shows that PU3 has folded C shaped structure in both bound and free state. PU3 thus forms acceptable lead for further development of purine scaffold drugs. PU3 attaches to N terminal domain via the following key interactions: at the top 2 methoxy group of phenyl ring attaches itself to lys 112 of N terminal domain, the 9 – N butyl chain occupy the lateral hydrophobic pocket and this chain represents one of the most important elements of selectivity of PU3 for HSp90 versus similar pockets, and at the base C6 amino group hydrogen bonds with asp93 – ser52.

Hsp90 Invades Outside

The cytosolic molecular chaperone HSP90 has now shown to promote maturation of the extracellular MMP2. By assisting MMP2, HSP90 promotes the migration of cancer cell through the extracellular protein meshwork. The HSP90 α isomer has the capability to integrate with membrane and invade the extracellular matrix. One such drug is DMAG-N-Oxide(DMO). It is an antagonist to cancer cell motility and invasive nature. It inhibits cell migration and invasion invitro and invivo but it does not inhibit intracellular HSP90 and its client proteins. Effect of DMO on cell motility and invasion is due to its ability to inhibit cell migration by interfering with leading edge actin polymerization and focal adhesion formation. DMO is exclusively cell impermeable inhibitor. It is confirmed by its inability to compete with intracellular HSP90 inhibitor 17AAG(tritium labelled) in intact SKBR3 cells. when T24 bladder carcinoma cells are incubated with DMO and GA, the positive control, and Akt & Raf-1 levels monitored it is seen that their levels are not affected in DMO treated cells. Also they have shown that DMO has antimetastatic properties independent of any growth inhibition properties. In a matrigel invasion assay DMO significantly and dose dependently inhibited T24 cell invasion at concentration of 0.1 to 1µM. this anti invasive property is comparable to its affinity for HSP90 (Kd = 0.6µM).

Novobiocin

Hsp90 is composed of well-conserved amino and carboxyl terminal region separated by a charged domain. Although the crystal structure of the amino terminal fragment of Hsp90 complexed with ATP, GA, or RD has recently been solved the crystal structure of the carboxyl terminal region of Hsp90 still remains undetermined. Structural requirements for NB binding to Hsp90 are unique. First, several point mutations in the amino terminus of the chaperone that abrogate both GA and RD binding either did not affect or actually augmented NB binding. Second, an amino terminal fragment comprising the GA/RD binding domain failed to bind to immobilized NB. the NB binding site to a region in the C-terminus of Hsp90 known to be important both for its dimerization and for the association of other co-chaperones.

Gamitrinibs

Hsp90 is responsible for folding of proteins in multiple signaling networks in tumorogenesis. Gamitrinibs are the class of drugs that specifically act on mitochondrial Hsp90. They induce sudden loss of membrane potential which is followed by membrane rupture and initiation of apoptosis. Also gamitrinibs are highly selective and does not affect normal cells.