Medicines Design Flashcards

Question

What are the stages of BER?

Answer 1

1. Glycosylase cuts the N-glycosidic bond (base DNA), leaving an AP site 2. AP endonuclease cuts the sugar phosphate backbone 3. DNA polymerase inserts correct base (short patch BER) or multiple bases (long patch BER) 4. 5' single strand end of break intermediate is processed to allow for litigation by DNA ligases

Answer 2

- environmental and endogenous alkylating agents generate cytotoxic and mutagenic lesions in DNA - where alkylation occurs on a ring nitrogen, base will become unstable and may cleave from sugar leading to ss break

Answer 3

Alkylating agents that covalently bind to DNA, forming cross-links that prevent DNA replication and transcription → leads to cell death, especially in rapidly dividing cells (like cancer).

Answer 4

1. Nitrogen mustard contains a bis-(2-chloroethyl)-amine group. 2. It undergoes intramolecular cyclisation, forming a highly reactive aziridinium ion. 3. This ion alkylates nucleophilic sites on DNA — especially the N7 position of guanine. 4. This can form: - Monoalkylated DNA - Intrastrand cross-links - Interstrand cross-links (the most cytotoxic) 5. This disrupts: - DNA structure - Replication - Transcription 6. Cell cycle arrest → apoptosis

Answer 5

- Guanine N7 position, most common, leads to depurination and strand breaks - Adenine N3 position, can disrupt base pairing and DNA stability

Answer 6

Small molecules that bind selectively to the minor groove of DNA, usually in AT-rich regions, without disrupting the helix structure. They fit into the narrow space of the minor groove, making non-covalent interactions

Answer 7

- Bind non-covalently to DNA, many have natural twist to fit into minor groove - Can block DNA-protein interactions (e.g., transcription factors, polymerases) - Some interfere with replication or transcription - Others can induce DNA cleavage when linked to reactive groups - Used to inhibit transcription or DNA replication in cancer cells

Answer 8

Early CPPIs were so cytotoxic they caused multi-organ failure in mice due to uncontrolled DNA alkylation. This led to their redesign into antibody-drug conjugates (ADCs) to safely harness their potency.

Answer 9

In hoogsteen, one base is rotated/flipped, the minor groove is compressed, major groove is widened and usually less stable, but important for flexibility Hoogsteen base pairs are found in distorted or damaged DNA

Answer 10

It’s essential for DNA unwinding and segregation, and inhibiting it causes lethal DNA breaks, especially in rapidly dividing cancer cells. It can: - Cut both strands of the DNA helix (double-stranded breaks) - Pass another segment of DNA through the break - Religate the strands — preventing tangling and supercoiling Blocking topo II traps it in a “cut-DNA” state → causing double-stranded DNA breaks that the cell can’t repair → cell death DNA damage sensors trigger apoptosis Cancer cells often have defective repair mechanisms, making them more vulnerable

Answer 11

Cardiotoxicity and secondary malignancies

Answer 12

Small, flat, aromatic molecules that slip between the base pairs of the DNA double helix, disrupting its structure and function. They don’t form covalent bonds, but physically insert themselves into the DNA stack

Answer 13

1. The drug intercalates into DNA at specific or random sequences. 2. This distorts the double helix: Unwinds DNA Prevents replication fork progression Blocks transcription 3. Many intercalators also: Inhibit topoisomerase II (double-strand breakage) Generate free radicals (→ oxidative damage)

Answer 14

Increases distance between base pairs Changes helical twist and supercoiling Causes DNA strand rigidity Can trap enzymes (like topoisomerases) in non-functional states Prevents RNA polymerase from initiating or continuing transcription All of which = cell cycle arrest and apoptosis

Answer 15

Myelosuppression Cardiotoxicity Mucositis, alopecia

Answer 16

A subclass of anthraquinone antibiotics, often more potent and clinically used in chemo Anthraquinone core + amino sugar Intercalates + inhibits topoisomerase II Generates free radicals → oxidative DNA/protein damage Cytotoxic, induces apoptosis via DNA damage

Answer 17

Telomerase is a ribonucleoprotein enzyme that adds repetitive DNA sequences to the ends of chromosomes (telomeres) to prevent them from shortening during cell division (which causes aging)

Answer 18

Telomerase binds to the 3' overhang of the telomere It extends the telomere using its RNA as a template DNA polymerase can then fill in the complementary strand Telomeres are preserved, not lost - highly active in embryonic stem cells and germ cells - Reactivated in cancer cells = immortality

Answer 19

- - ~85–90% of cancers show telomerase reactivation - Allows replicative immortality (one of the hallmarks of cancer) - Often involves upregulation of TERT expression - Potential drug target

Answer 20

- a mixture of cytotoxic glycopeptide abx isolated from strain of streptomyces verticillus and freely soluble in water - related basic glycopeptides which differ in the terminal amine substituent of bleomycin acid

Answer 21

- Used in conjunction with chemo for lymphoma, squamous carcinoma + germ cell tumors

Answer 22

- Bind to transition metals + O2 and can catalyse formation of ss+dsDNA lesions

Answer 23

- In vitro studies showed that a single molecule of bleomycin was powerful enough to generate lesions in DNA - Studies also indicated the whole molecule is greater than sum of its parts, each element essential for DNA cleavage

Answer 24

Outcomes are complex and cell-line/genotype dependent Extended cell-cycle arrest, apoptosis and mitotic cell death are most common

Answer 25

- Bleomycins are hydrophilic molecules unable to cross cell membrane - Studies indicate this may be due to +ve tail of BM

Answer 26

Metal binding region, linker region and Bithiazole tail

Answer 27

- bleomycins initiate ssDNA cleavage at pyrimidines to a guanine in a sequence specific fashion - secindary sight of cleavage can be either staggered or blunt based in DNA sequence - bleomycins cut a strand, regenerate and cut the second strand, to break DNA in half - cannot be fixed by NER or BER as no template

Answer 28

- Recombination can be initiated by a double-strand break (DSB) that may be caused by an endonuclease or a DNA-damaging agent. - The DNA is processed at the site of the break to yield regions of single-stranded DNA. - Rad51, assisted by replication protein A (RPA), coats the single-stranded DNA to form a filament that searches for homologous sequences and, when it finds them, initiates the formation of a joint molecule. - The break is repaired by DNA synthesis using the intact strands as templates

Answer 29

- cisplatin shown to have good properties back in 1965, first patient treated by 1971 - found when platinum inelectordes killed bugs in water - cisplatin for testicular, carboplatin for breast

Answer 30

- Patients more willing to try experimental drug

Answer 31

- Cancer can increase productions of glutathione, platins can react with glutathione and are deactivated (as with any electrophilic drug

Answer 32

- Platin activated with water, one Cl- swapped for water then associated with DNA and displaces - DNA platination

Answer 33

- increased platin delivery to tumors - liposomes, co-polymers - targeting tumors with leaky surfaces - intraperitoneal/bladder delivery - resistance modulators (TLK286, decitabine) - combination with biologics

Answer 34

- inactivation of VHL tsg - bevacizumab (first anti-VEGF therapy) - binds to and neutralises all biologically active VEGF - prevents growth and metastasis

Answer 35

- original assumed to be reacted but actually looking for sequences/bends etc - more to DNA than just a ladder - DNA 'breathes'

Answer 36

TNM T (tumor) 1-4 based on spread N (nodes) 0-1 whether spread to lymph nodes or not M (metastasis) 0-1 full spread or not Also uses classic staging 1-4

Answer 37

- prostate specific antigen made by prostate and is in the blood - if prosate enlarged, innflamed or infected PSA may be elevated

Answer 38

- Benign prostatic hyperplasia - low (then high) grade prostatic intraepithelial neoplasia (testosterone dependent) - metastatic prostate cancer (usually to bone) - androgen-independent cancer (mutation of AR gene)

Answer 39

- watchful waiting - active surveillance - surgery (often but major sfx) - hormone therapy (only when dependent) - chemo (hormone independent or relapse) - high-intensity focused ultrasound - external beam radiotherapy - permenant seed brachytherapy

Answer 40

- Testosterone binds to AR - This causes conformational change in AR - Complex translocates to nucleus, where it signals proliferation

Answer 41

- Flutamide (67) a non-steroidal anti-androgen, intially bacteriostatic, high heptotoxicity - (77) developed from flutamide, less hepatotoxicity, selcetive antagonist against AR - Bicalutamide (82) widely used in advanced PC, often combined with gonadotropin releasing hormone

Answer 42

- Nilutamide (2012) good in vivo efficacy, good pk profiles, high SS in brain - Apalutamide (2018) good in vivo efficacy, good pk profiles, low SS in brain - Darolutamide (2019) full antagonist, high AR affinity, short half-life

Answer 43

Binds to AR but doesn't cause conformational change, testosterone blocked from binding - Bic-AR complec is internalised but cannot translocate to nucleus - Antigen withdrawal syndrome - Bic has some agonist activity against mutant AR, removing bic slows progression

Answer 44

- H-bonds, hydrophobic interaction, Bicalutamide can also bind to AR - H-bonds etc structure not dissimilar

Answer 45

- AR signalling pathway alteration (typically AR gene amplification, more receptors gained) - AR mutation (can convert drugs from antagonist to agonist) - AR (splice) variants (splice variants arise, typically after castration or enzalutamide - metabolic changes (increased androgen synthesis can overwhelm inhibition) - lineage plasticity (cell-line becomes AR independent by large scale epigenetic changes)

Answer 46

- Cholesterol - pregnenolone - progesterone - testosterone - Three common enzymes involved (CYP17A1, 3ß-HSD, 17ß-HSD)

Answer 47

- Tetramer with 4 binding points - Oxidises compounds - Drugs inhibiting CYP17A1 (ketoconazole (non specific), abiraterone (selective of CYP17A1, acetate on prodrug, ligand to metal), progesterone (C=O forms H bind to ASM202))

Answer 48

1. Folate antagonists (methotrexate) 2. Pyramidine antagonists (5-fluorouracil) 3. Purine antagonists (thioguanine) 4. Sugar-modified (cytarabine)

Answer 49

- Folate aka vit B9 - Folic acid is synthetic, body converts to folate - Used as dietary supplement - Folate required for DNA synthesis + to metabolise amino acids - Dr Farber found use as essential for rapidly deividing cells and therefore its antagonist achieved remission

Answer 50

Enzymes - serine hydroxymethylytransferase (SHMT) (tetrahydrofolate > 5,10CH2-) - thymidylate synthetase (TSE) (5,10CH2- > dihydrofolate) - dihydrofolate > tetrahydrofolate)

Answer 51

Methotrexate - methyl in place of H, NH2 in place of carbonyl, oxidised to NH not N - analogue of dihydrafolate - binds to DHFR enzyme at binding site - very potent inhibitor of DHFR - too polar for passive diffusion - taken up by reduced folate carrier (RFC) - is polyglutamated to b eretained in cells

Answer 52

- Mutations to DHFR enzyme - MDR phenotype causing active reflux - RFC mutation

Answer 53

- Enter cells by passive diffusion - Pyrimethamine (inhibits DHFR of many species, mainly antibacterial) - Methylbenzoprim (very potent experimental lipophilic inhibitor of DHFR) - Piritrexin (potent lipophilic inhibitor of DHFR, active in several types of tumors) - Nolatrexed (inhibits CHFR and TS, active against liver carcinoma)

Answer 54

- 5,10-CH2-tetrahyrdrofolate - Pemetrexed - Raltitrexed

Answer 55

- Azacytidine is a nucleoside analogue used as an anti-cancer and epigenetic drug, primarily to treat certain leukemias and myelodysplastic syndromes which are characterised by hyper methylation and + silencing of multiple genes - DNA methylation is key epigenetic mechanism that silences genes without a change in coding sequence - Required for normal cellular development (not normal dynamic regulation of gene expression) - Cancer cells can exploit DNA methylation to counter TSGs - azacytidine was originally conceived as a cytotoxic agent, but subsequently found use as a hypomethylating agent

Answer 56

1. Epigenetic Regulation (at low doses) - metabolic conversionn into triphosphate - Incorporated into DNA - Inhibits DNA methyltransferases (DNMTs) — the enzymes responsible for adding methyl groups to cytosine bases. - This causes DNA hypomethylation, especially at tumor suppressor gene promoters, potentially reactivating silenced genes. - It's used to reverse abnormal epigenetic silencing found in cancers. 2. Cytotoxicity (at higher doses) - Incorporated into RNA - Disrupts RNA metabolism and function, impairing protein synthesis and causing cell stress and eventual apoptosis

Answer 57

The purine analogue (pro-drug) is taken up into the cell by nucleoside transporters. The analogue is converted into its monophosphate form - 6-MP poorly bioavailable as pro-drug undergoes extensive metabolism - 6-TIMP - ultimately forms 6-thioguanine nucleotide (6-TGN) Is then incorporated into DNA in place of guanine - DNA-TG can undergo random methylation, causing mismatching and ultimately strand breaking/cell death - complex metabolism gives huge variety in outcome

Answer 58

Cytarabine - converted to cytarabine trisphosphate - trisphosphate inhibits DNA polymerases as analogue of dCTP - some incorporation into DNA, making DNA non-functional Fludarabine - converted into trisphosphate - triphosphate inhibits DNA polymerase as analogue of dATP Gemcitabine - efficiently converted to trisphosphate F2dCTP and diphosphate F2dCDP -F2dCTP inhibits DNA polymerase as analogue of dCTP - 100x more potent than cytarabine

Answer 59

- Taken up into cell by hENT1 and hCNT3 transporter - Initial phosphorylation by dCK is rapid (rate-limiting step) - Further phosphorylation leads to the diphosphate (F2dCDP) + triphosphate (F2dCTP) Two mechanisms of cytotoxicity 1. Diphosphate inhibits RR (ribonuclease reductase) essential for making deoxynucleotides - RR is critical for cell management of pool of deoxy-nucleotides 2. The triphosphate is in corporated freely into DNA/RNA, causing masked-chain termination - in DNA, the next nucleotide can be added but the chain elongation stops

Answer 60

- DNA polymerase incorporated F2dCTP - another nucleotide is added - at this stage, polymerase is unable to continue so chain elongation stops - leads to apoptosis + other responses

Answer 61

- dFdCDP inhibits RNR, which is the key enzyme that converts ribonucleotides (like CDP) into deoxyribonucleotides (like dCTP) When RNR is inhibited: - The intracellular dCTP pool drops. - Meaning there’s less competition for dFdCTP to be incorporated into DNA. - As a result, more gemcitabine gets into DNA, intensifying its cytotoxic effects. - as dCTP is feedback inhibitor of dCK, as it decreases, dCK is upregulated - upregulation means more gemcitabine is phosphorylated into F2dCMP and F2dCDP, further reducing pool of CTP

Answer 62

Long slender filamentous tubes comprised of α-tubulin and β-tubulin heterodimers These subunits form dimers, which assemble into protofilaments. 13 protofilaments form a cylindrical microtubule. Connected by covalent bonds. +ve end associated with GTP cap and dynamics faster than -ve end but both can add/lose heterodimers Dynamism characterised by four factors - rate of MT growth - rate of MT shrinkage - frequency of transition from growth to shrinking 'catastrope' - frequency of transition from shrinking to growth 'rescue' - MT can also undergo treadmilling - units add to one end and leave from the other, allowing them to move

Answer 63

- Cell division (mitotic spindle) - Intercellular transport (like tracks for motor proteins) - Cell shape and structure (rigidity) - Cilia and flagella (drive movement in some cells)

Answer 64

- Both alpha and beta units have a GTP/GDP binding site at the +ve end - Within heterodimers, the alpha retains a GTP tightly bound (due to interections with beta end) and is non-exchangable - The beta end is accessible and hydrolysis of GTP to GDP occurs at the same time as addition to growing MT Growth phase ("polymerization") GTP-bound tubulin adds to the + (plus) end of the microtubule. As long as there’s a GTP-cap (GTP-tubulin at the tip), the microtubule keeps growing. Shrinkage phase ("catastrophe") Once GTP at the tip is hydrolyzed to GDP, the GTP cap is lost. GDP-bound tubulin is unstable → microtubule starts to depolymerize rapidly. This shrinkage can be reversed if a new GTP-cap forms, called a "rescue".

Answer 65

Interphase - microtubues turn over slowly - at the onset of mitosis, the entire network disassembles and is replaced by new population of spindle MTs that are 4-100x more dynamic Step 1 - Prometaphase - Nuclear envelope breaks down - Needs timely and correct attachments of chromosome kinetochores to the spindle - During this phase, MT 'hunt' by rapidly elongating + shrinking, probing the cytoplasm until chromosome kinetochore found - Grow and shrink up to 10 um until attachment found Step 2 - Early metaphase - Complex movement of chromosome - Congression - movement to equator to form metaphase plate Step 3 - Anaphase - Duplicated chromosomes separate - Chromosomes move towards two spindle poles and thus to form new duather cells Step 4 - Telophase - duplicate chromosomes have reached new spindle poles, cell is dividing

Answer 66

Microtubules are key to: - Forming the mitotic spindle during mitosis - Aligning and separating chromosomes - Maintaining cell structure and intracellular transport - microtubules seem to be favourite target for naturally acquiring (self-protective) toxic molecules produced by a wide range of plants and animals - overall success of such drugs leads to some argue microtubules are best target

Answer 67

- Surfaces of the globular part of tubulin has several binding sites allowing agents to bind - These microtubule targeting agents (MTAs) alter the dynamics - Ultimately lead to mitotic arrest and cell death - Six MTA binding sites known - Four on beta tubulin and 2 on alpha

Answer 68

- Paclitaxel - tetracyclic diterpenoid - Isolated from taxus brevifolia in 60s - Approved in 1992 by PDA for ovarian cancer - Currently produced semi-synthetically from european yew - Used in diverse cancer therapies - Sfx - myelosuppression, peripheral neuropathy

Answer 69

- Taxane pocket near the ML surface on 'inside' of tubulin - Acts to strengthen lateral contacts between adjacent protfilaments - leads to MT stabilisation - Paclitaxel possibly stabilises MT via an allosteric mechanism (preventing dimer compaction) - Microtubules become overly stable → no dynamic changes → defective spindle → mitotic arrest

Answer 70

- A typical MT, 10,000 tubulin units, requires 5,000 paclitaxel molecules - However just one paclitaxel per several hundred can reduce rate/extent of shortening by 50% - in HeLa cells (immortal), 8nM paclitaxel blocks mitosis 50%

Answer 71

- Laulimalide and peluroside derive from sea sponge - Similar to taxanes with differing mechanism - Bind to a different site, which is opposite side of ML surface - MT established by strengthening lateral contacts between proto-filaments - Evidence that the MSA can interect with both its host tubulin and adjacent tubulin

Answer 72

- Natural vinca alkaloids vincristine + vinblastine isolated from periwinkle in 50s - First gen achieved great clinical success - Notable against childhood leukaemia - Sfx - peripheral neuropathy MoA - Bind to +ve end of β-tubulin and prevent polymerization (microtubule formation) - Binding alter surface of +ve end, forming a wedge - this interferes with incorporation of new heterodimers - plus, vinca site ligands can casue tubulin oligomers, decreasing free tubulin pool - at high concs, MTs depolymerise - one/two ligands at the end of MT will reduce dynamics by 50% - Microtubules can't form a proper spindle → mitotic arrest

Answer 73

- Located on β-tubulin near the vinca alkaloid site. Binding here: - Blocks tubulin polymerization into microtubules - Destabilizes existing microtubules - Leads to mitotic arrest and apoptosis - Growing MT, maytansine binding pocket of +ve end accomodates the +ve end of the next alpha-tubulin unit - Incorporation of a ligand blocks this, impeding elongation - Used in ADCs

Answer 74

- Fully synthetic analogue of a marine product - A microtubule-targeting anticancer - Mainly binds with high affinity to +ve end of existing MT - Used in metastatic BC and unresectable liposarcoma - Shows non-mitotic effects e.g. vascular remodelling which reduces tumor hypoxia + decreased metstasis

Answer 75

Colchicine isolated from autmun crocus - used for gout - severe toxicity at anti-tumor doses - Binding site between alpha and β units, slow, practically irreversible - Binding to heterodimer stabilises in curved formation - Presence of colchine type ligand prevents addition of new tubulin dimers to microtubules, slowing microtubule polymerisation

Answer 76

why selected cancers - paclitaxel effective for ovarian, mammary and lung, ineffective for solid tumors - vinea alkaloid effective against haemato, usually ineffective for solid tumors modes of resistance - overexpression of membrane transport protein - MT associated protein - post translational modification - up/down regulation of different tubulin isotopes

Answer 77

- EGFR or TGR binds to extracellular domain of EGFR - Ligand-EGFR complex forms on assymetric dimer - Intracellular kinase domain autophosphorylases at Tyr992,1045 etc - SH2 domains of signalling proteins bind to phosphotyr + trigger signalling cascades - These trigger proliferation, migration, adhesion - EGFR upregulated in lung cancers, colon cancers, glioblastomas, head + neck tumors

Answer 78

- EGFR implicated overexpression or activating mutation in extra/intracellular domains - Mutations in extracellular domain can lead to constitutive (EGF-dependent) activity and are frequently found in glioblastoma - Mutations in kinase domain are found in sub set of NSCLCs (most common mutations L8585R) - These are driver mutations (confer growth advantage + so drive cancer) - The particular mutation occuring in tumor will determine the sensitivity of tumor to different drugs - Selecting drugs based on tumor mutation status is precision/personalised medicine - Targeted cancer therapy places a huge selective pressure on target to evolve, resistant mutations often occur

Answer 79

EGFR kinase domain found as active or inactive Inactive conformation has two hallmarks found in many inactive kinase structures - Alpha C helix rotated out away from kinase body, disrupting catalytic site - New helix at beginning of region of kinase known as activation loop, blocking substrate binding site Active conformation of kinase is catalytically competent - The activation loop is open, allowing access to ATP and substrate. - The alpha C-helix is rotated inward, forming part of the active site

Answer 80

Inactive conformation of EGFR is stabilised by hydrophobic cluster - No ligand bound to EGFR - Mutations that inactivate kinase activity Active conformation of EGFR is stabilised by - Ligand-induced dimerization - Certain activating mutations (e.g. L858R in NSCLC)

Answer 81

- EGFR is a crystallographic dimer, so is normally observed in active conformation - EGFR is only observed in inactive conformation (in presence of inhibitor lapatinib, and V948R mutation) - Crystallography gives us atomic resolution information on arrangment of protein in crystal but no info on conformational heterogenity or interconversion

Answer 82

- Gefitinib is an ATP-competitive inhibitor of EGFR kinase domain, licensed in Europe as first-line treatment of NSCLC with EGFR mutations - Cells with L858R EGFR mutation are 10-100x more sensitive to gefitinib compared with WT

Answer 83

- Structure of gefitnib bound to WT and L858R are identical and similar to EGFR bound to AMP-PNP - Binding data shows gefitinib binds tighter to L858R mutation than WT (can be rationalised in terms of preferential binding to active conformation, larger population of active conformation will result in tighter measured binding) - Cellular potency depends on competition between inhibitor and ATP can be estimated by ration of Kd inhibitor/Km1ATP, which is 100x more potent in L858R mutation)

Answer 84

Gatekeeper mutations are specific point mutations in the kinase domain of a protein that alter the shape or size of the ATP-binding pocket, making it difficult for tyrosine kinase inhibitors to bind, leading to drug resistance. - Targeted cancer therapy places selective pressure on target to evolve, and resistance mutations often occur - Eventually patients develop resistance to gefitinib, usually through mutation of structurally conserved residue called gate keeper to a bulkier residue - in EGFR, the gate keeper residue is T790 and the T790M mutation is most common

Answer 85

- Structure-based design - High-throughput screening including use of combinatorial libraries - Rational design

Answer 86

AMACR = α-methylacyl-CoA racemase Enzyme involved in fatty acid metabolism Overexpressed in prostate cancer, colorectal cancer, etc. Target for cancer diagnostics and therapeutics

Answer 87

LIC is a cloning technique that avoids using DNA ligase. It relies on: - Creating complementary single-stranded overhangs - These overhangs anneal precisely, enabling insertion into vectors - PCR using primers with LIC extensions - Template DNA removed by Dpn1 digestion - PCR product treated with DNA polymerase and ATP – converts LIC extension to stranded DNA - PCR product and plasmid (with complementary overhang) mixed at room temperature. Transformed into bacteria. - Bacteria containing plasmid selected using kanamycin resistance.

Answer 88

1. Gene Cloning of gene of interest into an expression vector 2. Transformation of the plasmid into a host cell: Rosetta2 (DE3) (contains additional tRNAs). 3. Expression Induction - DE3 component allows induction of protein expression using lac operon. - Bacteria grown in auto-induction media containing lactose. - Binding of lactose to the lac repressor causes protein to dissociate from DNA and transcription of AMACR sequence occurs. 4. Protein Purification - Protein has N-terminal sequence containing 6 His residues. - Make crude extract of proteins by lysing bacterial cells. - AMACR binds to Ni2+ ions in column by His residues. Other proteins are washed through. - Purified protein is eluted using imidazole-HCl (competes with His side-chains by chelating to Ni2+ ions). 5. Validation - confirm expression and identity using SDS-PAGE, the SDS unfolds proteins and makes them negatively charged so they migrate according to their size.

Answer 89

- High-throughput screening in 96 well plate using colorimetric substrate. - Drug is added as a DMSO solution to 30 µM final concentration. - Enzyme solution added and mixture is incubated together for 10 minutes (allows time for drug to bind to enzyme). - Substrate added to 40 µM final concentration (= Km value). These are called ‘balanced conditions’ because different types of inhibitor will exert their effects to a similar extent. - Activity is measured based on change in absorbance/ minute (easier to spot when inhibition is occurring). - Potential ‘hits’ are compared to positive and negative controls. Where inhibition is confirmed, potency is assessed using dose-response curves.

Answer 90

- Poly(ADP-ribose) [PAR] is the third nucleic acid in the mammalian cell - Polyanionic polymer built from ADP-ribose units derived from NAD+ - Usually built onto Glu side-chains in target proteins - Acts as a signal or scaffold in DNA damage response

Answer 91

PARP1 (Poly[ADP-ribose] Polymerase 1) is a DNA repair enzyme that plays a key role in the detection and repair of single-strand breaks in DNA. Enzyme mainly found in the nuclei of cells Transfers ADP-ribose units from NAD⁺ to proteins to make PAR

Answer 92

1. DNA Damage Sensing - Binds quickly to DNA single-strand breaks 2. Poly(ADP-ribosyl)ation (PARylation) - Uses NAD⁺ to build chains of poly(ADP-ribose) (PAR) - PAR chains are attached to target proteins - This recruits and organizes DNA repair machinery 3. Facilitates DNA Repair - Helps coordinate base excision repair (BER) - Temporarily relaxes chromatin to allow access to damaged sites 4. Auto-regulation - PARP1 auto-PARylates itself to regulate its activity and release from DNA 5. System must be returned to equilibirum - PARG is smashing chains apart, cleaved off of everything - Stopped PARP-1 will inhibit whole process

Answer 93

A PARP1/2/3 inhibitor Clinical trials started 2003, in combination with temozolomide Approved for ovarian, fallopian tube and peritoneal cancer Blocks DNA repair in cancer cells → DNA damage accumulation → cell death

Answer 94

A PARP1/2 inhibitor Phase 1-3 clinical trials, in combination and as single agent Triple negative breast cancer and NSCLC

Answer 95

A PARP1/2 inhibitor Approved as single agent for maintenance treatment of advanced ovarian, fallopian and peritoneal cancer after response to first line platinum therapy Blocks DNA repair in cancer cells → leads to cell death

Answer 96

- Highly potent PARP inhibitor used especially for BRCA1/2-mutated breast cancer - FDA approved in 2018 for metastatic breast cancer

Answer 97

- DNA damage occurs (radiation, alkylation etc - Single strand break develops - Can be repaired via BER etc - If not repaired then double strand break can form - Repair via BRCA1/2 - If patient lacks BRCA1/2 then the double strand repair does not proceed

Answer 98

Tumours need supply of O2 and other nutrients - angiogenesis Poor vascular structure - - disorganised network - vessel walls not well formed - leaky - high interstitial pressure Oxic region - Well oxygenated - Proliferating - Sensitive to radiotherapy & chemo Hypoxic region - Low oxygen concentration - Not proliferating - Relatively insensitive to radiotherapy & chemotherapy Radiotherapy requires O2  hypoxic tissue less sensitive to radiotherapy Radiosensitising drugs - Electron-affinic radiosensitisers (e.g. pimonidazole, etanidazole) - PARP-1 inhibitors Hypoxia-selective drugs - Nitroimidazoles (e.g. RB6145) - Mitomycin C - Tirapazamine

Answer 99

- Radiosensitising drugs are agents that make cancer cells more sensitive to radiation therapy. by interfering with DNA repair, oxygenation, or the cell cycle. - Molecular oxygen (O2) is a potent chemical radiosensitizer. This reflects its extreme electron-affinity – leading to the participation in the chemical reactions that lead to DNA damage after absorption energy from ionizing radiation. - Oxygen-mimetic radiosensitizers work in hypoxic cells by replacing oxygen in the chemical reactions that lead to DNA damage. - Metronidazole and similar compounds have been evaluated and are effective at radiosensitizing, but other issues remain.

Medicines Design Flashcards

(123 cards)