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Hemat/Onc EXAM 2
Hemat/Onc EXAM 2 - Crider Misc

Additional Pharmacology Flashcards




miscellaneous anticancer agents


inhibit proteasome and thereby increase levels of certain regulatory proteins that can kill cancer cells


azacytidine and decitabine

methyltransferase inhibitors actually block the hypermethylation of DNA

hypermethylation of DNA can result in the recruitment of certain trascriptional gene suppressors

by blocking hypermethylation of trascriptional suppressors, DNA methyltransferase inhibitors block the effect of gene silencing


vorinostat, romidepsin

histones are proteins that are found in nucleosomes

the tails of histones are subject to epigenetic modifications

acethylation of histone tails loosen chromatin and allow the approach and binding of certain transcription factors that lead to the expression of tumor suppressor and proapoptotic genes


numerous pharmacological effects

these compounds are known to have antiangiogenic activity and they can induce apoptosis
protein degradation essential for the cell
free supply of amino acids for protein synthesis

remove excess or unneeded proteins:
excess enzymes
transcription factors that are damaged or no longer required

structures responsible for protein degradation:

lysosomes - proteolytic action on extracellular proteins and transmembrane proteins

proteasomes - proteolytic action on endogenous proteins found in cytoplasm and nucleus
structure of the 26 S proteasome

the 26S proteasome consists of a catalytic 20S subunit that is composed of 28 subunits consisting of 4 concentric rings

there are 2 caps on each end of proteasome

the caps are composed of 17 proteins includine 6 ATPases

the role of the 26S proteasome is to degrade tagged proteins

these tagged proteins prevent the degradation of necessary proteins

in order for proteasome to degrade the protein the protein is first ubiquitinylated

ubiquitin is a protein

these tagged proteins are then degraded by proteasome to yield polypeptides

the small peptides are further degraded by the action of exopeptidases

the polyubiquitinylated protein is broken down by enzymes that split the individual ubiquitin subunits

in this manner, ubiquitin is regenerated
ubiquitin cycle and protein degradation

this is a further explanation of the action of the 26S proteasome

an activating enzyme first activated ubiquitin through its C-terminal glycine amino acid

this is followed by a conjugating enzyme in which additional ubiquitin subunits are attached

finally a ligating enzyme attaches the polyubiquitinylated chain to the protein to be degraded

the ubiquitin subunits are recycled through degrading enzyme to free the individual ubiquiting
significance of proteasome inhibition
blocking proteasome results in an accumulation of a various regulatory proteins, which lead to cell death by a variety of mechanisms (block cell cycle and apoptosis)

proteolytic action of proteasome (hydrolysis of peptide bond):


by inhibiting proteasome, levels of certain regulatory proteins rise

these proteins are involved in cell death by a variety of mechanisms

compounds that inhibit the action of proteasome alter the mechanism shown above

a key Thr amino acid is found on the N-terminal end of proteasome

the key to this reaction is the attack of the Thr OH group on the peptide bond of proteasome

the Thr residue is then attached to the enzyme (proteasome)

the final step is hydrolysis of the N-terminal ester bond and regeneration of the enzyme


bortezomib is a boronic acid that is formulated as a boronate with mannitol

in solution it reverts to the boronic acid

in the structure of bortezomib are 2 peptide bonds

bortezomib competes with the substrate for the active site of proteasome, resulting in a stable but reversible complex


inhibition of proteasome by bortezomib


boron readily accepts lone pair on oxygen through its empty p-orbital

note that the N terminal Thr OH attacks the boron

this results in a stable, but reversible complex in which the enzyme is inhibited


a crystal structure between bortezomib and the N terminal Thr on proteasome is shown

the peptide chain has significant H bonds at the active site of proteasome
epigenetic therapy of caner
epigenetic - alterations in gene expression that are not associated with changes in DNA sequences

DNA methylation

Histone tail modification

histones are proteins that are found associated with nucleosomes; their tails can undergo acetylation and thereby affect gene expression
DNA methyl transferase (DNMT)
methylate C5 of cytosine - mechanism of long term silencing of gene expression

methylation close to a transcription start site - block binding of transcription factors, recruitment of transcription repressors (methyl binding proteins)

goal of epigenetic chemotherapy:

prevent hypermethylation of DNA that could lead to long term silencing of genes crucial to normal cell function (usually tumor suppressor genes)

methylation of cytosine nucleotides by SAM and DNA methyl transferase:


the methyl group is introduced at the 5 position of the ring

the methyl group donor in this reaction is S-adenosyl methionine

certain nucleosides such as azacytidine can be incorporated into DNA or RNA and inhibit DNA methyl transferases

the net result is that hypermethylation of DNA and gene silencing is prevented

inhibitor of DNMT (azacitidine) - the only difference is the presence of N at position 5

methylation of cytosine by DNMT (SAM methyl group donor):


this shows the mechanism by which DNA methyl transferases methylate cytosine bases

the first step in the process is reaction of the SH group at the active site of the methyltransferases with the C-6 position of the cytosine base

the electron pair of this intermediate attacks the reactive methyl group on SAM

this methyl group is adjacent to a positively charged sulfur atom

thus, the cofactor is displaced and the methyl group is transferred to cytosine

the DNA methyl transferase is displaced by the loss of a H+ at position 5

this last step is ESSENTIAL in the action of the methyl transferase

in the absence of this proton at C5 the DNA methyl transferase is inhibited (bound to DNA) and is subject to degradation
nucleoside inhibitors of DNMT

incorporate into DNA (decitabine) and DNA/RNA (azacytadine)

form covalent intermediate complex with DNMT

DNMT trapped and cannot dissociate (degraded)

may also damage DNA by a DNMT-independent pathway


inhibition of DNMT by azacytidine

note the process is exactly as shown for the normal substrate

after methylation of N5 on azacytidine, there is no proton at the 5 position to be eliminated

thus, the enzyme is inhibited
inhibitors of histone deacetylase
chromatin (nucleus) complex of DNA and protein that make up chromosomes

nucleosomes - smallest structural unit of chromatin; 200 DNA base pairs and 8 DNA associated proteins (histones)

histones - N terminal ends protrude from nucleosomes and are subject to epigenetic changes that regulate gene expression

histone deacetylase plays an important role in how tightly chromatin is packed

inhibitors of this enzyme loosen chromatin and allow expression of key suppressor gene such as tumor suppressor gene
modification of histone: ACETYLATION

there is a balance between HAT and HDAC

the acetyltransferase produces the acetylation of Lys residues on the histone tails

this causes less association between the Lys residue and the negatively charged phosphate groups in the DNA backbone

the enzyme histone deacetylase catalyzes the hydroxylsis of the amide bond

once in the protonated form, the positively charged amino groups on the Lys side chain associated much more tightly with the negatively charged phosphate groups on the DNA backbone

actions of HAT and HDAC are opposed:

HAT RELAXES CHROMATIN permitting various transcription factors to interact with DNA - PROMOTES TRANSCRIPTION

HDAC CONDENSES CHROMATIN preventing access of various transcription factors - LEADS TO TRANSCRIPTION REPRESSION

HAT inactivity and HDAC overactivity has been associated with TUMORIGENESIS

thus, a histone deacetylase inhibitor can prevent the deacetylation of histone proteins

this will allow the approach of various transcription factors that can promote gene trascription (tumor suppressors and proapoptotic factors)

acetylation destabilizes the nucleosome

allows approach and binding of transcription factors

tumor suppressor and proapoptotic genes are expressed
active site of HDAC
active site of HDAC contains a Zn ion that facilitates the proteolytic cleavage of the acetylated lysine


at the active site of HDAC, there is a key Zn ion that complexes to Asp and His residues at the active site of the enzyme

the Zn activates a water molecule, allowing attack on the peptide bond of the acetylated Lys residue of the histone protein

thus, hydrolysis of the acetyl group is achieved
vorinostat - a histone deacetylase inhibitor


vorinostat is a hydroxamic acid that acts as an inhibitor of HDAC

key to its action is the ability of the hydroxamic acid to complex to the Zn ion at the active site of histone deacetylase

it thus inhibits the enzyme

hydroxamic acids are weak acids with pKas around 9

the acidic proton is actually associated with nitrogen rather than oxygen

the reason being is that the resulting anion is stabilized by resonance over the C=O group
romidepsin (IV prodrug) - inhibitor of HDAC

S-S is the key to its action

disulfide undergoes bioreduction and SH groups are generated

SH groups will strongly bind Zn; Zn is tied up and histone deacetylase is inhibited

around the outside there are a lot of H bond donors and acceptors that help stabilize the compound in the active site of the enzyme
thalidomide and lenalidomide


undergoes chiral inversion in the body (rare for this to happen)


can be hydrolyzed on either ring

thalidomide and its derivative presumably act at one or more sites of action:

1) direct antiapoptotic effect on tumor cells

2) inhibition adhesion of MM cells partly due to decreased production of IL-6

3) decreased angiogenesis due to inhibition of growth factors and cytokine release

4) enhanced T cell production of cytokines and natural killer cells


restricted distribution program

lower risk of ADRs compared to thalidomide

compared to thalidomide it is better absorbed b/c of the amino group increasing water solubility
protein kinase inhibitors
regulation of phosphorylation stages of signaling molecules

serine - threonine kinases

tyrosine kinases

kinases that are confined to cytoplasms

kinases that tranverse the cell membrane and function as ENZYME and RECEPTOR


signal transduction involving kinase linked receptors:

binding of a chemical messenger (variety of growth factors and growth hormones) which start the signaling cascade that involved various protein kinases

this process eventually controls transcription of genes in DNA leading to cell growth and cell division

it has been observed that many cancers have an excess of a specific growth hormone or growth factor or a specific protein kinase or protein kinase receptor

since these processes are eventually involved in signal transduction which leads to cell growth and division, it is reasonable to assume that protein kinase inhibition will be useful anticancer agents

growth factor signaling:

binding of agonist ligands to growth factor receptors causes receptor DIMERIZATION and ACTIVATION OF CYTOSOLIC PROTEIN KINASE DOMAINS

kinase activity allows each kinase to autophosphorylate the other enzyme kinase

leading to activation of multiple signaling pathways

ATP binding site on a protein kinase

all kinases use ATP as the phosphorylating agen

at the active site of the kinase there is a binding site for ATP and a binding site that binds the substrate (growth factors or growth hormones)

ATP binds loosely at the active site and there are regions that are unoccupied

depending on the kinase, there are amino acid differences that makes it possible to design a relative selective inhibitor of specific kinases

the purine base of ATP binds deep within the binding pocket and forms 3 important hydrogen bonds with the protein backbone of the kinase

the ribose sugar unit is bound in the sugar region and the triphosphate region lies in a cleft leading to the surface of the enzyme

there is also a hydrophobic pocket across from the purine binding site

at the entrance to this pocket is an amino acid residue termed the gatekeeper residue

in some kinases, the gatekeeper residue is large and block access to the pocket

this results in a small backpocket

in other cases, the gatekeeper residue is small and allows access to the backpocket (the backpocket is large)

all kinase inhibitors have something that mimics the purine ring of ATP

hing region on the kinase: key is there is H bonding between the backbone of the kinase and ATP (help orientate ATP or an inhibitor at the active site of the enzyme)

when the gatekeeper is Thr (smaller) it allows a larger back pocket
when the gatekeeper is phenylalanine (larger) it has a smaller backpocket

size of the hydrophobic back pocket determined by size of gatekeeper amino acid:


take home message - not all kinase inhibitors bind the same
almost all kinase inhibitors have a purine like moiety

kinase inhibitors can have very specific activity on kinases and will be specific for certain cancers
imatinib, dasatinib, nilotinib - inhibitors of BCR-ABL tyrosine kinase
BCR-ABL tyrosine kinase has been implicated in chronic myelogenous leukemia


circled are similar regions that can H bond to the linker region and put them in the right position like ATP

hydrophobic structures that fit in the hydrophobic pocket

solubilized by attaching a tail that binds to an allosteric site (doesn't have effect binding to the active site, but enhances solubility)

binding of imatinib to BCR-ABL kinase:


important interactions with the BCR-ABL tyrosine kinase:

1. notice that the pyrimidine ring is attached to the pyrimidine ring undergoes hydrogen bonding in the region of the kinase where the purine ring of ATP binds; hydrophobic binding pocket I remains unoccupied

2. the anilinogroup attached to the 2 position of the pyrimmidine ring undergoes H bonding with the Thr in the gatekeeper region

3. 2 methylphenyl group fits into hydrophobic pocket II

4. the N methylpiperazine ring fits into the allosteric site on the kinase; the N methyl piperazine ring is a water solubilizing group that enhances oral bioavailability
imatinib metabolism



predominately metabolized by CYP3A4 to the active des methyl metabolite
metabolism of lapatinib

reactive quinoneimine intermediate; this intermediate can undergo conjugation with glutathione to form various adducts
other kinase inhibitors

circled - mimic nucleophils that bind to the kinase via H-bonds
JAK/STAT pathway
multiple pathways of carcinogenesis and cancer metastasis

over activity will cause cell proliferation

binding of the ligand to the cytokine receptors causes their dimerization

JAKs then bind to the receptor and undergo transphosphorylation

this causes downstream phosphorylation of STATs (signal transducers and activators of transcription)

the activated STATs dimerize and translocate to the nucleus where they activate or repress gene transcription
ruxolitinib - inhibits JAK1, JAK2




metabolized by CYP3A4 to hydroxy and conjugated metabolites

most kinase inhibitors involve metabolism through CYP3A4 so the possibility of drug interactions is very high!
mTOR inhibitors: rapamycin analogues
activation of mTORC1 causes phosphorylation of 4EBP
this causes 4EBP to lose the ability to inhibit eukaryotic translation inhibition factor 4E to slow metabolism

when mTOR is activated it causes a signalling cascade; if you can inhibit mTORC1 it is effective against tumors





metabolized by esterases to rapamycin and also by demethylation



mTORC1 inhibitor

metabolized by CYP3A4
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