L9: Advances of HDACi in Cancer Therapeutics Flashcards
1
Q
What are histones?
A
- In eukaryotes, DNA packaged/wrapped around histones into nucleosomes which are composed of 147 base pairs of DNA and core histone proteins H2A, H2B, H3 and H4.
- Histone octamer: Made of 2 H2A-H2B dimers, and 1 H3-H4 tetramer
2
Q
How can target genes be activated? (With egs)
A
- Requires chromatin remodelling and histone modifications by sensing external cues -> DNA responds by post translation modification to histone -> acetylation of histones/adding more moieties (eg. amino acids)
- Histone 2A: If ubiquitin group is added on lysine molecule -> gene silencing
- Histone 2B: ubiquitin group added -> gene activation
- Proline on histone: can isomerise from cis (usual) to trans
- Proline 38 is close to lysine 36 on histone
- When proline is in cis position, histone tail shifts closer to DNA so chromatin is in a closed structure -> less access to regulatory area -> minimal gene expression, basal transcription
- When proline 38 is in trans, put methyl on lysine -> chromatin open -> regulatory protein has full access to regulate DNA -> active transcription
- Histone phosphorylation on Ser/Threonine/Tyrosine: phosphate is -ve charged and DNA backbone is -ve charged -> repulsion -> chromatin open -> activation
3
Q
What are the functions of HDAC and HAT and their effect on DNA?
A
- HAT and HDAC participate in regulation of gene expression by regulating the opening and closing of chromatin, mediated in addition to post translational stage of histone
- HDAC (overexpressed in cancer): Histone deacetylase, removal of acetyl group from lysine residues -> Compacted chromatin (as lysine is +ve without acetyl and DNA is -ve)
- HAT: Histone acetyltransferase, adding acetyl moiety onto lysine on histone 3 and 4 (H3/4) -> Relaxed chromatin (lysine is neutral and DNA is -ve) -> Regulatory molecules enter -> Increase gene expression/Allow for gene expression
4
Q
What are the different types of HATs?
A
- Type A HATs: Always in nucleus, acetylate nucleosomal histone within chromatin in nucleus -> regulate gene expression
- Type B HATs: Housekeeping role in cell, acetylating newly synthesized free histones in cytoplasm for transport into nucleus
5
Q
What are the different types of HDACs?
A
- Zn dependent (Zn moiety in active site) HDAC: Class I (1 active site), Class II (1-2 active sites), Class IV -> HDACi target Zn
- NAD+ dependent: Class III (Sir 2) -> Respond to redox status in cells, respond to NAD+ instead of Zn
6
Q
What is the effect of overexpression of HDAC in cancer cells?
A
- HDACs are involved in chromatin remodelling and gene regulation
- Aberrant HDAC activity is a hallmark of several cancers
- Overexpressed HDAC -> gene repression/silencing of TSG/proteins that regulate cell cycle -> preventing proteins (eg. pro apoptotic) from being made
- Hence, HDACi has been a focus for pharmaceutical anticancer R&D (good target for cancer cells since HDAC is overexpressed, and has more proteins so this can be targeted)
7
Q
What are some processes that HDACs impact on cancer biology?
A
- Gene expression
- Oncoprotein stability
- Cell migration
- Protein catabolism
- Cell cycle control (Hence, HDACi can induce cell cycle arrest, anti proliferative effect)
- Tumour immunogenicity (Increase in antigen presenting capacity; With HDACi, more immunoreactive, activate NK cell and subset of T cell -> killing tumour)
- Eg. BAX and cytochrome C, usually inactivated by HDAC
- With HDACi, BAX is induced and cell death cascade follows
- In metastasis, HDAC1/2 in tumour keeps expression of E catherin (epithelial cell marker) low and mesenchymal marker high -> Tumour can do EMT switch (first thing for invasion)
8
Q
How was HDACi discovered?
A
- Started with dimethyl sulfoxide (DMSO)
- A compound that caused terminal differentiation of murine erythroleukemia cells (As DMSO is synthesized to hexamethylene bisacetamide)
- FYI: Murine erythroleukemia cells gets differentiated after a few days with 2% DMSO -> hence DMSO needs to be low to see if drug is effective for cancer cells (should not be the effect of DMSO)
- Also cause chromatin remodelling
- Wanted to understand the mechanism of DMSO -> lead to interest in developing HDACi
- Developed SAHA (vorinostat), used to treat rare cancer cutaneous T-cell lymphoma
- Started the research on HDACi
- Antitumour action of compounds undergoing clinical trials now
9
Q
What are the different classes of HDACi?
A
- **Know that there are 4 classes, possible MCQ qn
- Short-chain fatty acid
- Hydroxamic acid
- Cyclic tetrapeptides
- Benzamides
10
Q
What are some examples of short chain fatty acids?
A
- Phenylbutyrate (One of the first HDACi tested in patients): Initially for urea cycle disorder but at high concentration, found to have HDACi activity
- Valporic acid (HDACi): Induces differentiation of promyelocyte leukemia cell and proliferation arrest and apoptosis of various leukemia cell line in vitro
- Used as anti-convulsant initially (anti-epileptic) for epilepsy/BPD but found that at high concentrations in vitro, has HDACi activity
11
Q
What are some examples of hydroxamic acid?
A
- First compound to be identified as HDACi
1) Suberoyl anilide hydroxamic acid (SAHA) - Target Zn in HDAC active site
- Helped define the model pharmacophore for future HDACi
- For Asian population, have less metabolising enzyme (for homozygous condition for lower metabolic variants of CYP2D6) -> SAHA stay longer in body -> dose varies/lower doses for Asians compared to clinical trial doses
- 2) Trichostatin A (TSA)
- Reversible inhibitor of HDAC (unstable, NOT prescribed to patients)
- Induce cell cycle arrest at G1, apoptosis and cellular differentiation
- Has some uses as anti-cancer drugs
- +ve control for experiment
12
Q
[Review again what are these drugs for] What are some examples of cyclic tetrapeptides?
A
1) Apicidin
- Ethyl ketone component
2) Depsipeptide
- Modulate the expression of genes
3) Trapoxin
**May ask MCQ qn: Which of the following is not a cyclic tetrapeptide?
13
Q
What are some examples of benzamides?
A
- 2 drugs undergoing trials
1) MS-275 - Currently studied for blood cancers
- Mice experiment and result?
- 2) CI-994 (originally for epilepsy)
- Realized that it has HDACi activity
- Mechanism for anti tumour activity unclear
- Causes accumulation of acetylated histones although is not able to inhibit HDAC activity in a direct fashion
14
Q
What makes HDACi a good target?
A
- High level expression of HDAC isoenzymes and a corresponding hypoacetylation of histones in cancer cells
- An attractive model for the antitumor action of HDAC inhibitors is that the increase in histone acetylation leads to the activation of transcription of a few genes where the expression causes the inhibition of tumor growth.
- In vitro and in vivo, HDAC inhibitors cause cell cycle arrest and differentiation of many tumor types.
- HDAC mediated deacetylation alters the transcriptional activity of nuclear transcription factors, including p53, E2F, c-Myc, nuclear factor B (NF-B), hypoxia-inducible factor 1 (HIF-1), ER and androgen receptor complexes
15
Q
What is the general MOA for HDACi?
A
- HDACi is toxic as monotherapy, hence usually used as combination therapy in hospital/clinical; done by lowering dosage for each drug to achieve same/better effect
- Normal scenario/phenotype: HSP90, a chaperone protein, bind to cancer protein X, holding the complex together and keeping cancer protein active. HDAC6 binds to lysine residue on HSP90, preventing HAT from acetylating lysine.
- Used for chronic myeloid leukemia (CML): HDAC6 inhibitor 7b induces BCR-ABL ubiquitination and downregulation and synergizes with imatinib
