Hsp90 inhibitors
Encyclopedia
Among heat shock proteins the focus on HSP90
has increased due to its involvement in several cellular phenomenon and more importantly in disease progression. Zhao and Houry, in 2005, demonstrated that HSP90 keeps the death proteins in an apoptosis
resistant state by direct association. Its wide range of functions results from the ability of HSP90 to chaperone several client proteins that play a central pathogenic role in human diseases including cancer, neurodegenerative diseases and viral infection. It has been shown by Hadden et al. that geldanamycin
directly binds to the ATP-binding pocket in the N-terminal domain of Hsp90 and, hence, blocks the binding of nucleotides to Hsp90. Analysis of the effects of Geldanamycin on steroid receptor activation indicates that the antibiotic blocks the chaperone cycle at the intermediate complex, preventing the release of the receptor from Hsp90 and, eventually, resulting in its degradation. Ana Martin et al reported that ewing’s sarcoma shows several deregulated autocrine loops mediating cell survival and proliferation. So their blockade is a promising therapeutic approach. Proteosome analysis revealed that Hsp90 is differentially expressed between ewing’s sarcoma cell lines, sensitive and resistant to specific IGF1R/KIT inhibitors. Ana martin et al. previously reported the in vitro IGF1R/KIT pathway blockade on ewing’s sarcoma cell lines and classified ewing’s sarcoma cell lines as resistant and sensitive to blockade of pathway. Inhibition of Hsp90 with 17AAG and siRNA
resulted in reduction of cell lines growth and survival. The inhibition of Hsp90 causes the proteosomal destruction of client proteins- Akt, KIT and IGF1R. This effect 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.
and Radicicol
which are the natural product inhibitors and are starting point for new approach.
HSP 90 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. These include steroid receptors Raf – 1, Akt, Met and Her 2. HSP90 has conserved unique pocket in N terminal region. It binds ATP & ADP and has weak ATPase
activity. This suggests that site acts as nucleotide or nucleotide ratio sensor. It is observed that nucleotides adopt unique C shaped bent shape when binding to this pocket. This is particularly unusual as nucleotides never adopt shape change in high affinity ATP/ADP sites. This also indicates that drugs that are developed should also have potential to adopt unique C shape conformation in order to bind the unique pocket. The rational for this unusual need i.e. to bend the structure, is based on thermo dynamical fact that the molecule which needs minimum structural changes to go from unbound to bound state should not pay much entropic penalties and binding would be reflected by enthalpic factors. Geldanamycin and radicicol 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. Geldanamycin and radicicol also inhibit mutated protein in cancer cells like P53
, Vsrc, BCR – ABL. It is worth to note that the normal counterparts are not inhibited. Geldanamycin is an effective HSP90 inhibitor still it cannot be used in vivo because of its high toxicity and liver damage ability. The speculation is that the benzoquinone functional group is responsible. The semi-synthetic derivative 17 AAG, with lower toxicity but same potency as geldanamycin is developed and is currently under clinical trials.
and etoposide
are widely used in the treatment of AML due to their ability to induce apoptosis in leukemic cells. The signaling pathways by which these drugs work are not completely understood, but direct effects as DNA damage, mitochondrial electron transport interference, generation of oxidizing radicals and proteasomal activation have been demonstrated or hypothesized. The 17-allylamino-17-demethoxygeldanamycin (17-AAG) derivative of GA is currently in clinical trial in cancer. Under normal conditions, Hsp90 acts on a wide range of client proteins and is essential for conformational maturation of numerous oncogenic signaling proteins, including protein kinases and ligand-regulated transcription factors.Hsp90 acts in a multiprotein complex with several co-chaperones. One of these, cochaperone p23, appears to stabilize Hsp90-complexes with steroid receptors and oncogenic tyrosine kinases. p23 also has chaperone activity on its own and is able to inhibit aggregation of denatured proteins in the absence of ATP. The ATP antagonist GA and its derivative 17AAG blocks p23 association with Hsp90, induces proteasomal degradation of survival signaling. Hsp90 client proteins, activates the apoptosis-associated doublestranded RNA-dependent protein kinase, PKR and promotes an apoptotic rather than a necrotic death type. p23 has increased expression in mammary carcinomas. In their study, Gausdal and colleagues found that anthracyclines and other chemotherapeutic drugs like cytarabine and etoposide, but not GA alone, induced caspase-dependent cleavage of p23. The cleavage could be catalyzed by either caspase-7 or caspase-3 and occurred at D142 or D145 in the C-terminal tail of p23 that is believed to be required for chaperone activity. The Hsp90 inhibitor GA was found to enhance caspase activation, p23 cleavage and apoptosis induced by anthracyclines. Finally they concluded that Hsp90, and consequently signaling mediated by client proteins in the Hsp90 multiprotein complex, may be targeted through p23 in chemotherapy-induced cell death in AML.
So based on these considerations and observations chiosis and colleagues theoretically designed following class of purines in which PU3 is the lead molecule.
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.
This discovery is exciting from a clinical perspective as it provides a novel therapeutic target and currently there are some drugs that are in clinical trials. 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 in vitro and in vivo 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. Also 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).
was also able to interact with Hsp90, and to disrupt Hsp90’s chaperone activity in a manner similar to GA and RD. Thus, cells exposed to novobiocin demonstrated rapid destabilization of various Hsp90 client proteins, including Raf-1, mutated p53, p60v-src, and p185erbB2. Unexpectedly, however, although NB antagonized Hsp90 binding to both solid phase GA and RD, NB’s Hsp90 binding domain appeared to be distinct from the amino terminal GA/RD/nucleotide binding domain. Further data demonstrated that the structural requirements for novobiocin 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. In contrast, analysis of progressively smaller carboxyl terminal Hsp90 peptides revealed the NB binding site to be contained within amino acids 538 to 728. Within this region, the removal of amino acids 657 – 677 severely compromised NB binding, and a synthetic peptide composed of amino acids 663 – 676 efficiently competed the binding of Hsp90 to immobilized novobiocin. Thus, these additional data localized 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. The binding of C-terminal Hsp90 fragments to NBSepharose was competed not only by excess novobiocin, but also by ATP. Additionally, the smallest C-terminal Hsp90 fragment that bound to immobilized NB also bound to ATPSepharose and this binding was competed by both novobiocin and ATP. Similar to its effects on NB binding, deletion of amino acids 657 – 677 from C-terminal Hsp90 fragments reduced binding to ATP-Sepharose by greater than 90 percent, while the synthetic peptide duplicating the amino acid sequence from residues 663 – 676 effectively blocked Hsp90 binding to immobilized ATP. Binding of Hsp90 C terminal fragments to ATP-Sepharose was competitively inhibited by both soluble ATP and NB, suggesting that the novobiocin binding domain which we earlier mapped to the carboxyl terminus of Hsp90 may overlap with a second nucleotide binding site on the chaperone. An Hsp90 fragment lacking the amino terminal 222 amino acids but containing the charged domain, failed to bind to ATPSepharose just as it failed to bind to NB-Sepharose, suggesting that the charged domain may regulate nucleotide access to the C-terminal site. In support of this hypothesis, removal of the charged domain restored ATP binding to C terminal Hsp90 peptide. Novobiocin will continue to be useful for dissection of the complex interplay between the different domains of Hsp90 and knowledge of how this interplay relates to chaperone function.
and pyrazole
based compounds. The recent rapid progress has built on a wealth of knowledge obtained with the natural product inhibitors and is a good example of the value of chemical biology studies in which the biological activity is identified first and then the molecular target is discovered by detailed biological studies. Current medicinal chemistry activities are focusing on the combined use of high throughput screening and structure-based design, coupled to the evaluation of the compounds in robust and mechanistically- informative biological assays. The next decade will be exciting in the HSP90 field as the clinical activity of the early geldanamycin-based drugs is rigorously evaluated while a series of synthetic small-molecule agents enter preclinical and clinical development. Particular areas of interest will include the potential for orally active HSP90 inhibitors and for the development of isoform-selective drugs that are targeted to particular members of the HSP90 family (DMAG –N-OXIDE). HSP90 inhibitors may also be evaluated in diseases other than cancer and where protein folding defects are involved in the disease pathology. It can be predicted that additional molecular chaperones will now be targeted for therapeutic intervention in cancer and other diseases. Furthermore, a portfolio of drugs can be envisaged that target various points in the protein quality control pathways of the malignant cell and other diseases states.
Hsp90
Hsp90 is a molecular chaperone and is one of the most abundant proteins expressed in cells. It is a member of the heat shock protein family, which is upregulated in response to stress...
has increased due to its involvement in several cellular phenomenon and more importantly in disease progression. Zhao and Houry, in 2005, demonstrated that HSP90 keeps the death proteins in an apoptosis
Apoptosis
Apoptosis is the process of programmed cell death that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation...
resistant state by direct association. Its wide range of functions results from the ability of HSP90 to chaperone several client proteins that play a central pathogenic role in human diseases including cancer, neurodegenerative diseases and viral infection. It has been shown by Hadden et al. that geldanamycin
Geldanamycin
Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 and inhibits its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.Geldanamycin induces the degradation of proteins...
directly binds to the ATP-binding pocket in the N-terminal domain of Hsp90 and, hence, blocks the binding of nucleotides to Hsp90. Analysis of the effects of Geldanamycin on steroid receptor activation indicates that the antibiotic blocks the chaperone cycle at the intermediate complex, preventing the release of the receptor from Hsp90 and, eventually, resulting in its degradation. Ana Martin et al reported that ewing’s sarcoma shows several deregulated autocrine loops mediating cell survival and proliferation. So their blockade is a promising therapeutic approach. Proteosome analysis revealed that Hsp90 is differentially expressed between ewing’s sarcoma cell lines, sensitive and resistant to specific IGF1R/KIT inhibitors. Ana martin et al. previously reported the in vitro IGF1R/KIT pathway blockade on ewing’s sarcoma cell lines and classified ewing’s sarcoma cell lines as resistant and sensitive to blockade of pathway. Inhibition of Hsp90 with 17AAG and siRNA
Sírna
Sírna Sáeglach , son of Dian mac Demal, son of Demal mac Rothechtaid, son of Rothechtaid mac Main, was, according to medieval Irish legend and historical tradition, a High King of Ireland...
resulted in reduction of cell lines growth and survival. The inhibition of Hsp90 causes the proteosomal destruction of client proteins- Akt, KIT and IGF1R. This effect 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 GeldanamycinGeldanamycin
Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 and inhibits its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.Geldanamycin induces the degradation of proteins...
and Radicicol
Radicicol
Radicicol, also known as monorden, is a natural product that binds to Hsp90 and alters its function. HSP90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, angiogenesis and oncogenesis.-Further reading: Review of the chemistry and...
which are the natural product inhibitors and are starting point for new approach.
HSP 90 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. These include steroid receptors Raf – 1, Akt, Met and Her 2. HSP90 has conserved unique pocket in N terminal region. It binds ATP & ADP and has weak ATPase
ATPase
ATPases are a class of enzymes that catalyze the decomposition of adenosine triphosphate into adenosine diphosphate and a free phosphate ion. This dephosphorylation reaction releases energy, which the enzyme harnesses to drive other chemical reactions that would not otherwise occur...
activity. This suggests that site acts as nucleotide or nucleotide ratio sensor. It is observed that nucleotides adopt unique C shaped bent shape when binding to this pocket. This is particularly unusual as nucleotides never adopt shape change in high affinity ATP/ADP sites. This also indicates that drugs that are developed should also have potential to adopt unique C shape conformation in order to bind the unique pocket. The rational for this unusual need i.e. to bend the structure, is based on thermo dynamical fact that the molecule which needs minimum structural changes to go from unbound to bound state should not pay much entropic penalties and binding would be reflected by enthalpic factors. Geldanamycin and radicicol 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. Geldanamycin and radicicol also inhibit mutated protein in cancer cells like P53
P53
p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
, Vsrc, BCR – ABL. It is worth to note that the normal counterparts are not inhibited. Geldanamycin is an effective HSP90 inhibitor still it cannot be used in vivo because of its high toxicity and liver damage ability. The speculation is that the benzoquinone functional group is responsible. The semi-synthetic derivative 17 AAG, with lower toxicity but same potency as geldanamycin is developed and is currently under clinical trials.
Geldanamycin derivative 17 AAG
17 AAG is the semi-synthetic derivative of natural product Geldanamycin. It is less toxic with same therapeutic potential as Geldanamycin. It is the first HSP90 inhibitor to be evaluated in clinical trials. Currently 17AAG is being evaluated as potent drug against AML. It is known that 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. Gene expression profiling of human colon cancer cell lines with 17AAG proves that Hsp90 client protein genes are not affected but the client proteins like hsc, keratin 8, keratin 18, akt, c-raf1 and caveolin-1 are deregulated resulting in inhibition of signal transduction. Acute myelogenous leukemia (AML) remains the most common form of leukemia in the adult and elderly population. Currently, anthracyclines, cytarabineCytarabine
Cytarabine, or cytosine arabinoside, is a chemotherapy agent used mainly in the treatment of cancers of white blood cells such as acute myeloid leukemia and non-Hodgkin lymphoma. It is also known as Ara-C...
and etoposide
Etoposide
Etoposide phosphate is an anti-cancer agent. It is known in the laboratory as a topoisomerase poison. Etoposide is often incorrectly referred to as a topoisomerase inhibitor in order to avoid using the term "poison" in a clinical setting...
are widely used in the treatment of AML due to their ability to induce apoptosis in leukemic cells. The signaling pathways by which these drugs work are not completely understood, but direct effects as DNA damage, mitochondrial electron transport interference, generation of oxidizing radicals and proteasomal activation have been demonstrated or hypothesized. The 17-allylamino-17-demethoxygeldanamycin (17-AAG) derivative of GA is currently in clinical trial in cancer. Under normal conditions, Hsp90 acts on a wide range of client proteins and is essential for conformational maturation of numerous oncogenic signaling proteins, including protein kinases and ligand-regulated transcription factors.Hsp90 acts in a multiprotein complex with several co-chaperones. One of these, cochaperone p23, appears to stabilize Hsp90-complexes with steroid receptors and oncogenic tyrosine kinases. p23 also has chaperone activity on its own and is able to inhibit aggregation of denatured proteins in the absence of ATP. The ATP antagonist GA and its derivative 17AAG blocks p23 association with Hsp90, induces proteasomal degradation of survival signaling. Hsp90 client proteins, activates the apoptosis-associated doublestranded RNA-dependent protein kinase, PKR and promotes an apoptotic rather than a necrotic death type. p23 has increased expression in mammary carcinomas. In their study, Gausdal and colleagues found that anthracyclines and other chemotherapeutic drugs like cytarabine and etoposide, but not GA alone, induced caspase-dependent cleavage of p23. The cleavage could be catalyzed by either caspase-7 or caspase-3 and occurred at D142 or D145 in the C-terminal tail of p23 that is believed to be required for chaperone activity. The Hsp90 inhibitor GA was found to enhance caspase activation, p23 cleavage and apoptosis induced by anthracyclines. Finally they concluded that Hsp90, and consequently signaling mediated by client proteins in the Hsp90 multiprotein complex, may be targeted through p23 in chemotherapy-induced cell death in AML.
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, phenomenologically relevant scaffolds can be constructed. Random in vitro screening of library of small purine-related molecules led to identification and screening of more than 60000 compounds that have inhibition potency. Chiosis and colleagues reported the novel class of HSP90 inhibitors using rational design. The important factors considered in this rational design are- Key interaction between inhibitor and Asp 93/ser 52 and lys 112/lys 58 at the base and top of the pocket respectively.
- Occupancy by inhibitor of hydrophobic pocket laying midway in the binding site and constituted by met98, val 150, leu 107, leu 103, phe 138 and val 186 is essential for affinity and selectivity.
- Molecules should have superior affinity to HSP90 as compared to natural nucleotides.
- Since many proteins depend on purine containing ligands for their function, derivatives of purine skeleton should have bioactivity, cell permeability and solubility.
So based on these considerations and observations chiosis and colleagues theoretically designed following class of purines in which PU3 is the lead molecule.
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. In fact this chain represents one of the most important elements of selectivity of PU3 for HSp90 versus similar pockets.
- At the base C6 amino group hydrogen bonds with asp93 – ser52
HSP 90 invades outside
It is known that metalloproteases are required for the invasive nature of cancer cells. Surprisingly 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. Using fluorophore assisted light in activation technique, it is concluded that only the HSP90 α isomer has the capability to integrate with membrane and invade the extracellular matrix. The standard cytosolic form of HSP90 that is found in normal cells does not have membrane signal sequence, so it was surprising to find HSP90 in oxygen rich extracellular matrix and ATP deficient plasma membrane core. The genome analysis revealed the mystery by pointing to alternative transcript of HSP90α gene with two extra exons preceding to the standard first exon. Closer inspection reveals that translation initiation at an ATG within the second exon would produce extended HSP90 with a predicted N terminal signal peptide. After its cleavage the secreted HSP90 would encompass the entire known HSP90 sequence with only a 3K terminal extension.This discovery is exciting from a clinical perspective as it provides a novel therapeutic target and currently there are some drugs that are in clinical trials. 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 in vitro and in vivo 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. Also 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
Functional analysis has revealed that 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,[18] the crystal structure of the carboxyl terminal region of Hsp90 still remains undetermined. However, biochemical characterization of this portion of the chaperone suggests that a complex interaction between carboxyl terminal and amino terminal domains is a critical regulatory component of Hsp90 function. Using Hsp90 binding to ATP-Sepharose as a readout, we included NB in a natural product screen of inhibitors of the Hsp90-ATP interaction. Indeed, novobiocinNovobiocin
Novobiocin, also known as albamycin or cathomycin, is an aminocoumarin antibiotic that is produced by the actinomycete Streptomyces niveus, which has recently been identified as a subjective synonym for S. spheroides a member of the order Actinobacteria . Other aminocoumarin antibiotics include...
was also able to interact with Hsp90, and to disrupt Hsp90’s chaperone activity in a manner similar to GA and RD. Thus, cells exposed to novobiocin demonstrated rapid destabilization of various Hsp90 client proteins, including Raf-1, mutated p53, p60v-src, and p185erbB2. Unexpectedly, however, although NB antagonized Hsp90 binding to both solid phase GA and RD, NB’s Hsp90 binding domain appeared to be distinct from the amino terminal GA/RD/nucleotide binding domain. Further data demonstrated that the structural requirements for novobiocin 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. In contrast, analysis of progressively smaller carboxyl terminal Hsp90 peptides revealed the NB binding site to be contained within amino acids 538 to 728. Within this region, the removal of amino acids 657 – 677 severely compromised NB binding, and a synthetic peptide composed of amino acids 663 – 676 efficiently competed the binding of Hsp90 to immobilized novobiocin. Thus, these additional data localized 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. The binding of C-terminal Hsp90 fragments to NBSepharose was competed not only by excess novobiocin, but also by ATP. Additionally, the smallest C-terminal Hsp90 fragment that bound to immobilized NB also bound to ATPSepharose and this binding was competed by both novobiocin and ATP. Similar to its effects on NB binding, deletion of amino acids 657 – 677 from C-terminal Hsp90 fragments reduced binding to ATP-Sepharose by greater than 90 percent, while the synthetic peptide duplicating the amino acid sequence from residues 663 – 676 effectively blocked Hsp90 binding to immobilized ATP. Binding of Hsp90 C terminal fragments to ATP-Sepharose was competitively inhibited by both soluble ATP and NB, suggesting that the novobiocin binding domain which we earlier mapped to the carboxyl terminus of Hsp90 may overlap with a second nucleotide binding site on the chaperone. An Hsp90 fragment lacking the amino terminal 222 amino acids but containing the charged domain, failed to bind to ATPSepharose just as it failed to bind to NB-Sepharose, suggesting that the charged domain may regulate nucleotide access to the C-terminal site. In support of this hypothesis, removal of the charged domain restored ATP binding to C terminal Hsp90 peptide. Novobiocin will continue to be useful for dissection of the complex interplay between the different domains of Hsp90 and knowledge of how this interplay relates to chaperone function.
Gamitrinibs
Targeting networks of signaling pathways instead of single pathway is effective way for cancer treatment. Hsp90 is responsible for folding of proteins in multiple signaling networks in tumorogenesis. Recently it is seen that mitochondrial Hsp90 is involved in complex signaling pathway that prevents initiation of induced apoptosis. 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.Future perspective
HSP90 is gaining increasing importance as a cancer target, in large part because of the potential for combinatorial targeting of multiple oncogenic protein pathways and biological effects. The good tolerability seen with the first-in-class drug 17-AAG has encouraged many biotechnology and large pharma companies to enter the field. The ability to demonstrate proof of concept for target modulation in patients has also been encouraging, as has the early evidence of clinical activity in melanoma 17-AAG is now in Phase II studies as a single agent and combination studies with cytotoxic and other agents such as the proteasome inhibitor bortezomib are also underway. Improved formulations for parenteral use are also being evaluated in the clinic. Radicicol-based inhibitors have not entered clinical development. Following on from the initial proof of concept studies with the natural product agents, considerable progress has been made in the preclinical development of small molecule, synthetic inhibitors, as exemplified by the purinePurine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
and pyrazole
Pyrazole
Pyrazole refers both to the class of simple aromatic ring organic compounds of the heterocyclic diazole series characterized by a 5-membered ring structure composed of three carbon atoms and two nitrogen atoms in adjacent positions, and to the unsubstituted parent compound...
based compounds. The recent rapid progress has built on a wealth of knowledge obtained with the natural product inhibitors and is a good example of the value of chemical biology studies in which the biological activity is identified first and then the molecular target is discovered by detailed biological studies. Current medicinal chemistry activities are focusing on the combined use of high throughput screening and structure-based design, coupled to the evaluation of the compounds in robust and mechanistically- informative biological assays. The next decade will be exciting in the HSP90 field as the clinical activity of the early geldanamycin-based drugs is rigorously evaluated while a series of synthetic small-molecule agents enter preclinical and clinical development. Particular areas of interest will include the potential for orally active HSP90 inhibitors and for the development of isoform-selective drugs that are targeted to particular members of the HSP90 family (DMAG –N-OXIDE). HSP90 inhibitors may also be evaluated in diseases other than cancer and where protein folding defects are involved in the disease pathology. It can be predicted that additional molecular chaperones will now be targeted for therapeutic intervention in cancer and other diseases. Furthermore, a portfolio of drugs can be envisaged that target various points in the protein quality control pathways of the malignant cell and other diseases states.