Contents
Hsp90
Molecular Chaperone Inhibitors:Opportunities and Challenges
Guest
Editor: Paul Workman
Overview: Translating Hsp90 Biology into
Hsp90 Drugs Pp.297-300
Paul
Workman
Structure and Functional Relationships of
Hsp90 Pp.301-323
Chrisostomos
Prodromou and Laurence H. Pearl
Natural Product Origins of Hsp90 Inhibitors Pp.325-330
Yoshimasa
Uehara
Genes and Proteins Governing the Cellular
Sensitivity to HSP90 Inhibitors: A Mechanistic Perspective Pp.331-341
Alison
Maloney, Paul A. Clarke and Paul Workman
The C-Terminal Half of Heat Shock Protein 90 Represents
a Second Site for Pharmacologic Intervention in Chaperone Function Pp.343-347
Monica
G. Marcu and Leonard M. Neckers
The Stress Response: Implications for the
Clinical Development of Hsp90 Inhibitors Pp.349-358
Luke Whitesell, Rochelle Bagatell and Ryan Falsey
Development of Radicicol Analogues Pp.359-369
Shiro
Soga, Yukimasa Shiotsu, Shiro Akinaga
and Sreenath V. Sharma
Development of Purine-Scaffold Small Molecule
Inhibitors of Hsp90
Pp.371-376
Gabriela Chiosis, Brian Lucas, Henri Huezo, David Solit, Andrea Basso and Neal Rosen
Clinical Development of 17-Allylamino,
17-Demethoxygeldanamycin
Pp.377-383
Edward A. Sausville, Joseph E. Tomaszewski and Percy Ivy
The Clinical Applications of Heat Shock
Protein Inhibitors in Cancer – Present and Future Pp.385-390
Udai Banerji, Ian Judson and Paul Workman
[Back to top] Overview: Translating Hsp90 Biology into Hsp90 Drugs
Paul Workman
The Hsp90 molecular
chaperone has emerged as one of the most exciting targets for cancer drug
development. Hsp90 is overexpressed in many malignancies, very likely as a
result of the stress that is induced both by the hostile cancer
microenvironment and also by the mutation and abberant expression of
oncoproteins. A particularly attractive feature of Hsp90 as a cancer drug
target is that it is required for the conformational stability and function of
a wide range of oncogenic ‘client’ proteins, including c-Raf-1, Cdk4, ErbB2,
mutant p53, c-Met, Polo-1 and telomerase hTERT. Inhibition of Hsp90 should
therefore block multiple mission critical oncogenic pathways in the cancer
cell, leading to inhibition of all the hallmark traits of malignancy. This
combinatorial blockade of oncogenic targets should give rise to board spectrum
antitumour activity across multiple cancer types. The ‘druggability’ of Hsp90
was confirmed by the discovery that the natural products geldanamycin and
radicicol, which have anticancer activity, exert their biological effects by
inhibiting the essential ATPase activity associated with the N-terminal domain
of the protein. The first-inclass Hsp90 inhibitor has entered clinical trial
and provided proof of concept that Hsp90 can be inhibited and clinical benefit
seen at non-toxic doses. Further development is underway and a related analogue
17DMAG also shows promise in preclinical models. In addition, novel Hsp90
inhibitors have been identified using methods such as high throughput screening
and x-ray crystallography. The opportunities and challenges involved in
translating the fast moving biology of Hsp90 into patient benefit is discussed.
[Back to top] Structure and Functional Relationships of
Hsp90
Chrisostomos
Prodromou and Laurence H. Pearl
Understanding the
mode of action of Hsp90 requires that molecular detail of its interactions with
client proteins and co-chaperones are known. The structure determination of the
N-terminal domain of Hsp90/Hsp90b, proof that
it is an ATPase, that this activity is regulated and the identification of
cochaperones that facilitate Hsp90 function were landmarks towards
understanding conformational changes in Hsp90 brought about by ATP,
co-chaperones and client proteins. Sti1 and Cdc37/p50, which associate with
early Hsp90 complexes, were shown to be inhibitors of Hsp90 ATPase activity and
therefore promote its ‘open’ state, whereas Sba1/p23, which associates with
mature complexes, inhibits ATPase activity and stabilises the ‘closed’ state.
The isolation and characterisation of Aha1, the only known strong activator of
Hsp90 ATPase activity, which promotes the ‘closed’ state of Hsp90, will also be
of major importance in understanding Hsp90 function. The structure
determination of the middle region of Hsp90 has shed further light on the
complex ATP-cycle of Hsp90, identifying a catalytic loop, with key residues
that are essential for ATP hydrolysis. These studies, together with biochemical
ones, suggest that ATP hydrolysis, is dependent on a complex rate-limiting
step, involving N-terminal dimerization and association of the middle region,
and therefore the catalytic loop, of Hsp90 with the N-terminal domains. The
structure of the middle region of Hsp90 will also accelerate our understanding
of client protein interactions since this region is implicated in their
recognition and in particular their active-site openings.
[Back to top] Natural Product
Origins of Hsp90 Inhibitors
Yoshimasa
Uehara
The currently used
Hsp90 inhibitors, geldanamycin, herbimycin A and radicicol, were isolated many
years ago from Streptomyces and fungi originally for their antiprotozoal
activity, herbicidal activity and antifungal activity, respectively. In the mid
1980s, it was found that the benzoquinone ansamycin antibiotics (herbimycin A,
geldanamycin, and macbecin) reversed v-Src transformed cells to normal
phenotypes, and Bcr-abl was subsequently suggested to be the molecular target
for the treatment of chronic myelogenous leukemia through a study using
herbimycin A for its selective antioncogenic activity. In 1994, these
ansamycins were found to bind to Hsp90 and to cause the degradation of client
proteins including Src kinases; further efforts to develop anticancer drugs were
made using geldanamycin analogs, and 17AAG was chosen as the best candidate for
clinical trials. The number of novel natural products isolated from microbial
origins is continuing to increase and is doubling every 10 years. Thus,
screening of bioactive substances from natural origins, using assays including
defined targets, and developing leads toward drugs via optimized derivatization
is a conventional but still promising strategy for drug discovery and
development.
[Back to top] Genes and Proteins Governing the Cellular
Sensitivity to HSP90 Inhibitors: A Mechanistic Perspective
Alison Maloney, Paul A. Clarke and Paul Workman
HSP90 inhibitors
such as 17AAG have the major therapeutic advantage that they exert downstream
inhibitory effects on multiple oncogenic client proteins. They therefore block
several mission critical cancercausing pathways and have the potential to
modulate all of the hallmark biological features of malignancy. Consistent with
this combinatorial anti-oncogenic profile, 17AAG exhibits broad-spectrum
antitumour activity against cultured cancer cell lines and in vivo animal
models. However, there are clear differences in sensitivity between various
cancer cell lines and it is quite possible that some tumour types or individual
patients will be more responsive in the clinic than others. We describe the
methods used to investigate the genes and proteins involved in the mechanism of
action of HSP90 inhibitors and discuss the significance of these for cellular sensitivity.
Methods used involve the conventional cell and molecular biology techniques,
together with the more recent application of high throughput global
technologies such as gene expression microarrays and proteomics. Selected
examples that seem to play a role in sensitivity to HSP90 inhibitors are
highlighted and the potential relevance to the response of cancer patients is
discussed. Important determinants of response include: 1) Dependence upon key
HSP90 client proteins such as ERBB2, steroid hormone receptors and AKT/PKB; 2)
Levels of HSP90 family members and co-chaperones, such as HSP70 and AHA1; and
3) expression of various cell cycle and apoptotic regulators. In the case of
17AAG, metabolic enzymes such as NQO1 and membrane efflux pumps are also important
for sensitivity.
[Back to top] The C-Terminal Half of Heat Shock Protein 90
Represents a Second Site for Pharmacologic Intervention in Chaperone Function
Monica
G. Marcu and Leonard M. Neckers
The molecular
chaperone heat shock protein 90 (Hsp90) is required for stability and function
of multiple mutated, chimeric, and over-expressed signaling proteins that
promote cancer cell growth and/or survival. It is also critical for the
function of many normally expressed proteins, including protein kinases,
steroid receptors and other transcription factors, and it may protect the cell
from incapacitating or deleterious mutations. The recent identification of a
nucleotide binding pocket within the first 220 amino acids of the protein,
together with the discovery that at least two structurally distinct classes of
antibiotic can replace nucleotide at this site and alter chaperone activity,
has deservedly focused attention on Hsp90’s amino terminus as an important
regulator of function. However, data continue to accumulate pointing to the
Cterminal half of the chaperone as an equally important regulator of activity,
and small molecules that bind to this portion of Hsp90 have been identified.
[Back to top] The Stress Response: Implications for the
Clinical Development of Hsp90 Inhibitors
Luke
Whitesell, Rochelle Bagatell and Ryan Falsey
In their role as
molecular chaperones, heat shock proteins serve as central integrators of
protein homeostasis within cells. As part of this function, they guide the
folding, assembly, intracellular disposition and proteolytic turnover of many
key regulators of cell growth, differentiation and survival. Not surprisingly
then, heat shock proteins are over expressed in many types of cancer, and
induction of the stress response may actually be required for cells to tolerate
the genetic disarray characteristic of malignant transformation. Regulation of
heat shock protein levels via the stress response is complex, but recent data
indicate that the molecular chaperone Hsp90 plays a key role. Specifically,
Hsp90 inhibitors alter the multi-chaperone complexes associated with Heat Shock
Factor 1 (HSF1), the dominant transcription factor controlling induction of the
stress response, and stimulate HSF1-activated heat shock gene expression.
Induction of this heat shock response has now emerged as an important
consideration in the further clinical development of Hsp90 inhibitors for
several reasons. First, tumors in which the stress response is compromised
appear particularly sensitive to Hsp90 inhibition. Second, induction of the
stress response by Hsp90 inhibitors provides a sensitive pharmacodynamic
endpoint with which to monitor drug action in individual patients. Third, Hsp90
inhibitors display important therapeutic interactions with both conventional
DNA-targeted chemotherapeutics and newer molecularly targeted agents. These
interactions are, at least in part, due to modulation of the stress response by
these drugs. Lastly, stress response induction by Hsp90 inhibitors may have
therapeutic benefits in non-neoplastic disorders such as heart disease, stroke
and neurodegenerative diseases. These benefits are just beginning to be
explored.
[Back to top] Development of Radicicol Analogues
Shiro
Soga, Yukimasa Shiotsu, Shiro Akinaga
and Sreenath V. Sharma
Radicicol, a
macrocyclic antibiotic produced by fungi, was originally isolated many years
ago, and was described as tyrosine
kinase inhibitor. We also rediscovered radicicol as an inhibitor of signal
transduction of oncogene products, such as K-ras and v-Src, using yeast and
mammalian cell-based assays. In a study of mechanisms of action, it was
revealed that radicicol depletes the Hsp90 client signaling molecules in cells,
and thus inhibit the signal transduction pathway. In addition, direct binding
of radicicol to the Nterminal ATP/ADP binding site of Hsp90 was shown, and thus
radicicol has been recognized as a structurally unique antibiotic that binds
and inhibits the molecular chaperone Hsp90. Although radicicol itself has
little or no activity in animals because of instability in animals, its oxime
derivatives showed potent antitumor activities against human tumor xenograft
models. Hsp90 client proteins were depleted and apoptosis was induced in the
tumor specimen treated with radicicol oxime derivatives. Taken together, these
results suggest that the antitumor activity of radicicol oxime derivatives is
mediated by binding to Hsp90 and destabilization of Hsp90 client proteins in
the tumor. Among Hsp90 clients, we focused on ErbB2 and Bcr-Abl as examples of
important targets of Hsp90 inhibitors. Radicicol oxime showed potent antitumor
activity against ER negative/ErbB2 overexpressing breast cancer and Bcr-Abl
expressing CML. Putative mechanisms of action and future directions of
radicicol oxime against these kinds of tumor are discussed.
[Back to top] Development of Purine-Scaffold Small Molecule
Inhibitors of Hsp90
Gabriela
Chiosis, Brian Lucas, Henri Huezo, David Solit, Andrea Basso and Neal Rosen
The Hsp90
chaperones play a key role in regulating the physiology of cells exposed to
environmental stress and in maintaining the malignant phenotype in tumor cells.
Agents that interfere with the function of the chaperone may thus be beneficial
in the treatment of cancers. The ansamycins (geldanamycin and herbimycin) and
the unrelated natural product radicicol were found to bind to the N-terminal
pocket of Hsp90 and inhibit its function. However, translation of these
compounds to the clinic was impeded by stability and hepatoxicity issues.
17AAG, a derivative of geldanamycin, was found to be less hepatotoxic and is
currently undergoing Phase I clinical trial. Unfortunately, 17AAG is insoluble,
difficult to formulate and it is not yet clear if therapeutically effective
doses can be administered without escalating non-Hsp90 associated toxicities.
Additionally, for reasons not yet completely understood, a subset of tumor
cells are insensitive to the action of the drug. The development of novel
agents that lack the drawbacks of the natural products is thus necessary. Here
we present an overview of such efforts with focus on a new class of
purine-scaffold Hsp90 inhibitors developed by rational design.
[Back to top] Clinical Development of 17-Allylamino,
17-Demethoxygeldanamycin
Edward
A. Sausville, Joseph E. Tomaszewski and
Percy Ivy
17-allylamino,
17-demethoxygeldanamycin (17AAG; NSC 330507) is the first modulator of heat
shock protein 90 (Hsp90) to enter clinical trials. Hsp90 serves a chaperone
role to properly fold and deliver client proteins to appropriate intracellular
locations. Interest in Hsp90 modulators for the experimental therapeutics of
cancer has arisen based on pre-clinical evaluations suggesting that Hsp90
client proteins regulate signaling pathways critical to the molecular economy
of many types of tumors, including oncogene signaling, cyclin-dependent kinase
activation, steroid hormone receptors, and mediators of invasion and
metastasis. Thus, Hsp90-directed agents could affect molecules upon which
tumors depend for their proliferation and survival. Initial clinical studies
have therefore sought to incorporate assessment of these endpoints into initial
clinical evaluations. Three schedules of administration have been supported for
initial evaluation in Phase I studies sponsored by the National Cancer
Institute (NCI) or supported by NCI and sponsored by Cancer Research UK. In the
daily times five schedule, a recommended Phase II dose (RPTD) of 40 mg/m2
has been reached, while once weekly or three of four weekly schedules are
defining RPTDs of 295 and 308 mg/m2. Toxicity is tolerable and appears
dominated by hepatic, gastrointestinal, and constitutional symptoms.
Concentrations of drug at peak of ~1700-3000 nM are concordant with
concentrations predictive of useful outcomes in pre-clinical model systems.
Evidence of modulation of Hsp90 partner molecules has been obtained in both
surrogate and some tumor compartments. These very early results encourage
additional clinical evaluations of 17AAG and related molecules.
[Back to top] The Clinical Applications of Heat Shock
Protein Inhibitors in Cancer – Present and Future
Udai
Banerji, Ian Judson and Paul Workman
The potential
clinical applications of the prototype first-in-class Hsp90 inhibitor 17AAG and
other emerging Hsp90 drugs are very exciting. Rigorously planned and executed
clinical trials, incorporating measurement of appropriate biomarkers and
pharmocodynamic endpoints are critical for selecting the optimal dose and
schedule. A detailed understanding of the molecular mode of action of Hsp90
inhibitors alongside the elucidation of the molecular pathology of individual
cancers will help us to identify tumour types and individual patients that will
benefit most from treatment. Careful in vitro and in vivo experiments are
needed to choose the most potentially advantageous combination studies. It is
important to construct a pharmacologic audit trail linking molecular biomarkers
and pharmacokinetic and pharmacodynamic parameters to tumour response
endpoints. Phase I clinical studies with 17AAG have shown that the drug can be
given at does that are well tolerated and that also achieve active
pharmacokinetic exposures and elicit molecular signatures of gene and protein
expression that are indicative of Hsp90 inhibition. Furthermore, examples of disease
stabilization have been documented, consistent with the generally cytostatic
responses that are seen in animal models. Selecting tumour types for Phase II
clinical trials must involve balancing 1) our knowledge of molecular response
determinants, such as the expression of and dependence upon key client proteins
and 2) more pragmatic evidence of antitumour activity in the relevant
preclinical models. Examples of likely disease targets include chronic myeloid
leukaemia, melanoma, breast, ovarian, brain, thyroid, colorectal and prostate
cancer.