Promising anticancer agents : HSP 90 inhibitors

Abstract-
INTRODUCTION :
Cancer is a disease caused by the uncontrolled, abnormal growth of cells in different anatomic
sites. In 2018, it was predicted that the worldwide cancer burden would rise to 18.1 million new
cases and 9.6 million deaths. Anticancer compounds, often known as chemotherapeutic
medicines, have gained much interest in recent cancer research. These medicines work through
various biological processes in targeting cells at various stages of the cell's life cycle. One of the
most significant roadblocks to developing anticancer drugs is that traditional chemotherapy
affects normal cells and cancer cells, resulting in substantial side effects. Types of cancer

The overexpression of HSP90 occurs in patients with cancer, and the HSP90 triggers unstable
harmful kinase functions, which enhance carcinogenesis. Therefore, the development of potent
HSP90 inhibitors with high selectivity and specificity becomes very imperative.

HSP 90 inhibitors
Based on the crystal structures of yeast Hsp90 and Grp94 .Hsp90 isoform in mammalian
endoplasmic reticulum Hsp90 exists as a homodimer, each monomer consisting of three highly
conserved domains: an N-terminal ATP-binding domain (25 kDa), a middle domain (35 kDa)
and a C-terminal dimerization domain (12 kDa) In eukaryotes, the N-terminal and middle
domains are connected by a charged linker. The N-terminus of Hsp90 contains a specific ATP
binding pocket. The major role of the middle domain is to discriminate various types of client
proteins to adjust the molecular chaperone for proper substrate activation The C-terminal
dimerization domain strengthens the weak association between the two N-terminal domains of
the Hsp90 dimer. The C-terminal domain of eukaryotic Hsp90 has a conserved pentapeptide
(MEEVD) implicated in binding to the tetratricopeptide repeat (TPR) domain of cochaperones,
such as Hop (Hsp organizing protein) and Sti1 (stress-inducible protein 1, yeast homologue of
Hop.

Geldanamycin is a benzoquinone ansamycin antibiotic that manifests anti-cancer activity

through the inhibition of HSP90-chaperone function. The HSP90 molecular chaperone is
expressed at high levels in a wide variety of human cancers including melanoma, leukemia, and
cancers in colon, prostate, lung, and breast. In cancer cells dependent upon mutated and/or
over-expressed oncogene proteins, HSP90 is thought to have a critical role in regulating the
stability, folding, and activity of HSP90-associated proteins, so-called “client proteins”. These
client proteins include the growth-stimulating proteins and kinases that support malignant
transformation. Recently, oncogenic activating BRAF mutants have been identified in variety of
cancers where constitutive activation of the MEK/ERK MAPK signaling pathway is the key for
tumorigenesis, and they have been shown to be client proteins for HSP90. Accordingly, HSP90
inhibition can suppress certain cancer-causing client proteins and therefore represents an
important therapeutic target

HEAT SHOCK PROEIN:-

HSP90 is a dimer comprised of monomers with an overall structure consisting of three main
conserved domains known as the N-terminal domain (NTD), C-terminal domain (CTD), and
middle domain (MD] These domains are connected by linkers that allow the domains to
rearrange as HSP90 undergoes conformational changes . In eukaryotes, there exists a charged
linker domain that connects the N-terminal and middle domains This charged linker domain
varies in length and amino acid sequence composition.

Each of these domains takes on a specific function, and they are necessary for avoiding the
accumulation of damaged or misfolded proteins, especially in aging . The NTD is the binding
site for ATP and is thus called the nucleotide-binding site . Hence, the NTD is needed for HSP90
ATPase activity, necessary for the chaperone cycle and binding of client proteins to the HSP90
chaperone . The CTD plays a significant role in protein dimerization. It has two main sites: one
for calmodulin binding and another for homodimerization of HSP90 . The CTD also has a
nucleotide-binding site that opens when the NTD is occupied, thereby acting as an allosteric
regulator of the N-terminal ATPase activity . The CTD contains special motifs, MEEVD, or KDEL,
which differ based on the HSP90 isoform and its location within the cell—either in the
cytoplasm or the ER . The MD functions in the binding of substrates. The MD also regulates
HSP90 ATPase activity, as seen when the substrate binds to the MD, causing an increase in
ATPase activity . Although known to be present in only eukaryotic HSP90, the charged linker
domain is essential for the function, interaction, and flexibility of the HSP90 chaperone .

Dimerization is necessary for the proper function of HSP90. Specifically, mammalian HSP90 is a
phosphorylated dimer with monomers, each containing 2–3 covalently-bound phosphate
molecules. In addition, despite having an ATP binding site in the NTD, HSP90 has not only
ATPase activity but also GTPase activity.

HSP90 functions in the hallmarks of cancer:-

Being abundantly expressed in cancer, HSP90s promote growth and survival of tumor cells by
regulating a wide range of processes. Here, we will explore HSP90 involvement in the
hallmarks of cancer – a model of multi-step cancer development established by
Hanahan and Weinberg.

HSP90s

1Regulation of apoptosis,ferroptosis,necroptosis,autophagy,pyroptosis
2P53 Kinase regulation

3P53CDK

4Telomerase stabilization Senescence

5Cytokine production

6EMT

7Tumor antigen presentation

Regulationof immune
checkpoints

Enhancing the therapeutic potential of HSP90 Inhibition:

Despite the recognition of Hsp90 as an important anti-cancer target with pleiotropic effects on
many oncogenic client proteins, Hsp90 inhibitors have not to date demonstrated their
predicted level of clinical efficacy. Some of the key objectives that, if achieved, would further
realize the full therapeutic potential of Hsp90 inhibition.

1) Molecular stratification: not all Hsp90 clients are equally important

2) Minimizing the heat shock response.
Client Fuction
Kinases
AKT(PKB) Mitogen signaling
3)
BRAF Optimizing dosing and schedule through pharmacodynamic biomarkers.
Mitogen signaling
4) Dissecting and exploiting the complex EGF
ERBB2 molecular and cellular response to Hsp90
receptor
inhibition
BCR-ABL Constitutively active Tyr kinase
5) Use of combinotorial strategies.

SRC Constitutively active Tyr kinase

CDK4 Cell cycle control
JAK1 and/or KAJ2 Cytokine signaling
HCK Immune response
Transcription factors
P53 Tumor suppressor protein
HIF-1 alpha Angiogenesis
OCT4 Embryonic development
STAT2,STAT3,STAT5 Cytokine signaling
Steroid hormone receptors
Progesteron receptor Response to progesterone
Estrogen receptor Response to estrogen
E3 ubiquitin ligases
MDM2 p53 degradation
UHRF1 DNA methylation
Others
TERT Telomere maintenance
RAD51 and/or RAD52 DNA repair

HSP90 Inhibitor
 HSP 90Inhibitor

Inhibitors of HSP90 Cell surface

Agents that HSP90
the N-terminal Inhibitors HSP90
bind to the C- Inhibitors
ATP Binding Targeting Inhibitors
terminal/midd Targeting Co-
chaperon site Client/HSP90
le Domain of
/HSP90 Associations
HSP90-
Coomarin Interaction
Derivative

Natural Product Synthetic Small

and Their Molecule and
Peptide Derivative

Purine Scaffold
Geldanamycin and
Series
analogs

Radicolol and Resorcyclic

analogs Pyrazole

HSP 90 INHIBITORS CLASSIFICATION

1st generation :-

Natural : Geldanamycin ,Radicolol

 Derivative: 17-AAG , 17- DMAG , IPI -504, WK 88-1

2nd generation :-

Synthetic Radicolol-based Derivative: NVP – AUY922 , ATI3387 , STA-9090,GRP94 .

Purine and purine –like analogue: CNF -2024, Debio 0932(formerly CUDC -305,CUR-
0374441),PU-H71 .

Tropane derivative – XL 888

OTHERS –

Dihydroindazolone derivatives –snx -5422

Selective cytosolic HSP90 Inhibitors- TAS -166

Vaccine –gp96 HSP – Peptide complex .

MECHANISM: –
1 .Geldanamycin and its Derivatives

Geldanamycin MOA :-

Geldanamycin (GA) and its derivatives have been reported to possess multiple
pharmacological properties- antitumoral properties, inhibition of angiogenesis and
metastasis of diseases such as multiple myeloma , as well as breast or prostate cancer
GA have been identified from Streptomyces hygrocopicusin 1970 as tyrosine kinase
inhibitor.

MOA:-

Geldanamycin as depletor of Hsp90. Primarily Hsp90 client protein binds to

Hsp40/Hsp70 early chaperone complex.

Subsequently undergoes Hsp90 dimer- binding thanks to HOP- Hsp90/Hsp70

Organizing Protein forming intermediate complex.

ATP molecule hydrolysis results in dissociation of HOP and Hsp40/Hsp70, and

successively in association of Hsp90 with its co-chaperones, client protein.
 Geldanamycin binds to N-terminal ATP-binding site of Hsp90, perturbs formation of
mature complex and leads to proteasomal degradation by E3 ubiquitin ligase like CHIP
(Carboxy-terminus of Hsp70 Interacting Protein).

2.Radicolol:-

Radicicol is a macrocyclic natural antibiotic initially isolated from the fungus Monocillium
nordinii and Monosporium bonorden (. Like GA, radicicol was also shown to compete
with nucleotide for N-terminal ATP pocket of Hsp90 . Similarly, this interaction between
radicicol and Hsp90 constrains the chaperone in its ADP-bound conformation, leading
to destabilization of Hsp90 client proteins

3.PU-H71: Compound exhibit broad spectrum of anticancer activity.

MOA:-

PU-H71 high Selectivity for ATP binding sites of HSP-90.

Unique Conformational landscape of HSP90 In tumor allows PU-H71 to engage with its
ATP binding sites Conformational changes in HSP90 structure, stabilizes client protein
HSP90 Complex.

Degradation of oncogenic proteins(Client protein involved in key Pathways:

MPPK/PI3K/JAK&STAT.)that are essential for tumor cell survival &proliferation.

By disturpting the stability of this proteins, PU-H71 effectively presenting a targeted

therapeutic strategy against cancer.

4. Debio 0932(CUDC-305) : Oral HSP90 inhibitor having high bioavailability.

Structure: Similar to PU-H71,Nitrogen at 3rd position is replaced by Carbon &Iodine is
replaced by dimethylamine. ↓↓↓

Moa: CUDC-305 have high affinity for HSP90 alpha/ beta, Promote degradation of
HSP-90 Client proteins Binds strongly to Hsp90 drivers EGFR,FLT3,HER2.

Durable inhibition of multiple oncoproteins &induction of apoptosis. Degradation of

receptor tyrosine kinases and downstream signaling molecules of the PI3K/AKT&
RAF/MEK/ERK Pathways simultaneously.

It can cross blood brain barrier. have selective retention in tumor. Have dose dependent
antitumor activity hence mainly preferred for the t/t of brain cancer.

5.XL888

Is a heat shock protein-90 (HSP90) inhibitor.

↓
decreases in the growth of all the cell lines with no significant difference in IC 50 values
observed between the naive and resistance pairs of cell

↓
Treatment of all of the vemurafenib resistant cell lines with XL888 (300 nM) induces
high levels (>66%) of apoptosis,

↓
caspase-3 cleavage and loss of mitochondrial membrane potential (TMRM) in every
cell line tested.

↓
Treatment of cell lines that are naïve, intrinsically resistant and with acquired
vemurafenib resistance with XL888 (300 nM)

↓
leads to robust time-dependent increases in the expression of HSP70 isoform 1
(HSP71)
6 Ganetespib is a second-generation HSP90 inhibitor that belongs to the class of radicicol
derivatives, which arey composed of the resorcinol moiety. Unlike first-generation inhibitors,
ganetespib does not contain the benzoquinone moiety, which has been associated with
hepatotoxicity. This synthetic small-molecule inhibitor is resorcinol-based and non-
geldanamycin, and contains a triazolone moiety In vitro, ganetespib demonstrates strong
cytotoxicity in several hematological and solid tumor cell lines, including those that express
mutant kinases that make a tyrosine kinase inhibitor for tiny molecules resistant. Ganetespib
demonstrates continuous activity even after brief exposure times , in addition to quickly
degrading recognized HSP90 client proteins . Ganetespib cytotoxicity in these cell lines is
mostly caused by an irreversible commitment to apoptosis, most likely after impacts to the cell
cycle and growth arrest .

In vivo, ganetespib exhibits strong anticancer properties by substantial growth inhibition or

regression in hematological tumor models and solid tumor models, respectively. Compared to
geldanamycin analogs, ganetespib does not cause the dose-limiting liver damage that has been
reported. The ocular toxicity associated with NVP-AUY922 and SNX-522 has not been observed
with ganetespib because ganetespib is quickly removed from the retinal tissues and does not
build up in the rat eye .