WEEK 3.pdf

NUCLEAR RECEPTORS AS DRUG TARGETS: PHARMACODYNAMICS PHARMACODYNAMICS ◦ Study of biochemical and physiological effects of drugs on organs and cells TERMINOLOGY ◦ Receptors: macromolecule that ligand binds to producing biological effects ⁃ Needs to produce biological effect to be called recept...

NUCLEAR RECEPTORS AS DRUG TARGETS: PHARMACODYNAMICS PHARMACODYNAMICS ◦ Study of biochemical and physiological effects of drugs on organs and cells TERMINOLOGY ◦ Receptors: macromolecule that ligand binds to producing biological effects ⁃ Needs to produce biological effect to be called receptor ◦ Ligands: subtance that binds with receptors, based on conformation of ligand and receptor, lock and key ◦ Agonist: drugs bind to receptors to ACTIVATE biological effect ◦ Antagonist: drugs bind to receptors to NOT ACTIVATE, blocks bio. effect ⁃ If BOTH (agonist & antagonist) bind to receptor, the biological effect is smaller LOCK AND KEYS ◦ Nuclear Receptor Ligands: small MW, highly lipophilic (nonpolar) ⁃ Highly lipophilic ligands are needed b/c the receptors are located IN CELL, so ligands need to be able to pass plasma membrane ◦ Ligand - key, receptor - lock ◦ Nuclear Receptor Structure (NRS) ⁃ 4 Domains: ⁃ N terminus: AF-1(activation function region), variable (not conserved) ⁃ DNA binding domain (DBD): zinc finger motif involved phosphorylation conserved ⁃ Hinge: receptor bent @ this location, involved in transport, has nuclear localization signal (helps locate receptor to nucleus) ⁃ C terminus (lock): ligand binding domain (LBD, where ligand binds), involved in dimerization ⁃ Folded ⁃ Heat Shock Proteins(HSP): when there is no ligand, HSPs surrounds NRS, bends/distorts DBD so zinc fingers to not interect w/ DNA response elements, receptor is INACTIVE when surrounded by HSPs  ⁃ Ligand binding: ligand binds to LBD = HSPs leaves thus activating receptor & removes distortion & zinc fingers can interact with DNA ⁃ Dimerization: another NRS w/ ligand binded on LBD will come & dimerize with the first NRS w/ ligand, and then both will translocate into the nucleus, and in nucleus there is a hormone response element (HRE) ⁃ HRE: base pair sequence specific for the hormone (dimerized NRSs) ◦ Nuclear Receptor Types ⁃ Type 1: Cytosolic Receptors (intracellular, located on cytoplasm) ⁃ Steroid receptors: estrogen receptors, adgrogen recoetros ⁃ Type 2: Nuclear Receptors (Within nucleus on the DNA itself) ⁃ Retinoid X receptor, thyroid hormone receptor TYPE 1: CYTOSOLIC RECEPTORS ◦ Signaling Mechanism of Action - HOMOdimers, uses HSPs ⁃ Binds to soluble receptor protein in cytoplasm or inside nucleus ⁃ Then dimers (same receptors) binds to DNA base pairs by zinc fingers TYPE 2: NUCLEAR RECEPTORS ◦ Signaling Mechanism of Action - HETEROdimers, uses corepressor ⁃ Ligand binds directly to DNA proteins b/c receptors are on DNA protein  ⁃ Binds as heterodimers (two different receptor) to DNA ⁃ When heterodimers are NOT bound to ligands they are bound to corepressors ⁃ When ligand binds = corepressors leave and coactivator + RNA polymerase attaches to heterodimer complex ⁃ Intiates transcription resulting in protein being made NUCLEAR RECEPTORS AS DRUG TARGETS: SELECTIVE RECEPTOR MODULATORS SELECTIVE RECEPTOR MODULATOR (SRM) ◦ Drug that has different effects depending on tissues types ⁃ Agonist in some tissus, antagonist in other ⁃ AKA: Tissue selective drugs, mixed agonist/antagonists ◦ Includes: SERM, SPRM, SARM ⁃ Drugs being sold in USA are SERMS SEX HORMONE RECEPTORS ◦ Testosterone: produced from leydig cells in testes for men and ovaries for women ⁃ Bound to carrier protein in the plasma, goes through type 1 signaling pathway  ⁃ Conversion to more protent androgen can happen (dihydrotestosterone) ⁃ dihydrotestosterone mediates many androgen actions ⁃ Dihydrotestosterone and estradiol is the metabolized version of testosterone ⁃ Dihydrotestosterone reduced by 5alpha-reductase enzyme ⁃ Estradiol aromatized by CYP19 (aromatase) enzyme ⁃ Tesosterone and dihydrotestosterone binds to androgen receptors ⁃ Estradiol binds to estrogen receptor ⁃ Androgen and estrogen receptors are found in most tissues ⁃ These are type 1 nuclear receptors signaling pathway (cytosolic receptor) SERMS (SELECTIVE ESTROGEN RECEPTOR MODULATORS) ◦ Goal: Create compounds that act on different estrogen receptors and are tissue specific ◦ Estrogenic effects on bone and liver ◦ ANTI estrogenic effects in breasts ◦ -ifen: common spelling in SERMs, uses Class 1 nuclear signaling pathway ◦ Only class of drug approved by USA market PHARMACOLOGICAL ACTIONS OF SERMS (REVIEW) ◦ Tamoxifen: treatment option of breast cancer, prevention of breast cancer ⁃ Acts as a antagonist in breast tissue ⁃ Acts agonist in other parts of the body ⁃ Mostly has agonist and antagonist effects on ER alpha (ERa) ⁃ No effects on ER beta (ERb) ◦ Raloxifene: treatment option and prevention of osteoporosis in POSTmenopausal women ⁃ ERa: agonist and antagonist effects ⁃ ERb: antagonist effects ◦ BOTH (tamoxifen & raloxifene): ⁃ Agonist : bone, liver, blood ⁃ tamoxifen affects uterus ⁃ Antagonists (blocking receptor) : breast, periphery, brain, raloxifene affects uterus ⁃ Blocking estrogen effects can cause same things you see in menopause ◦ Clomiphene Citrate ⁃ Fetility pill ⁃ Used as treatment and prevention of breast cancer ⁃ Due to toxic effects it was abandoned ⁃ binds to ERa & ERb ⁃ ER a effects dependent on estrogen levels ⁃ Agonist if estrogen is low ⁃ Antagonist if estrogen is normal or high ⁃ ERb antagonist ⁃ Blocks ERs in the hypothalamus/negative feedback functions ⁃ Causing increases FSH & LH secretory pulse ⁃ Negative feedback: ⁃ Brain signals when hormones are low to the pituitary as GnRH ⁃ GnRH acts on pituitary (specifically anterior pituitary) and causes release FSH & LH ⁃ FSH & LH acts on testes in men and ovaries in women ⁃ Acting on gonads realease testosterone in men and estradiol & progesterone in women ⁃ Hormones go back to the body/plasma and feedback to ghe pituitary and hypothalamus (ensure body is blanaCed w/ hormones) ⁃ With SERM ⁃ Serms acts in hypothalamus, pituitary, gonads  ⁃ serms block estrogen receptors in those tissues ⁃ Blocking receptors/antagonist causes no signal activation ⁃ body think not enough sex hormones are made so the body will amplify the negative feedback function causing increase in FSH & LH ⁃ Adverse effects of amplification: polycystic ovaries, multiple pregnancies, ovarian hyper stimulation syndrome SARM (SELECTIVE ANDROGEN RECEPTOR MODULATOR) ◦ Goal: high specifity for androgen receptor, needs to be tissue selective ◦ Testosterone will be 1:1 ratio of anabolic and adrogenic ◦ Perfect SARM will have high anabolic and low androgenic ratio ◦ Androgeneic vs Anabolic ⁃ Androgenic ⁃ Related to male characteristic development, promote secondary male characteristics (facial hair), used for androgen therapy ⁃ Anabolic ⁃ resembles testosterone for muscle growth, tissue growth (bone & muscle), treatment for anemia/muscle wasting/puberty growth/ osteoporosis ◦ SARM is still being developed ⁃ 1st SARM in 1988 ⁃ SARMS doesnt affect prostrate so it could be a good treatment option for prostrate cancer ◦ SARM act @ androgen receptors ⁃ Class 1 nuclear receptors (cytosolic) ⁃ Depending on tissue results in different co regulators being activated thus modualting gene transcription ⁃ Each SARM comples will gave diff. configuration b/c each SARM will have different conformations ⁃ Having diff. conformation will result in diff. coregulators or diff. Activity at androgen response element ⁃ Each tissue will have its own pattern of androgen receptor expression, coregulatory protein levels - ALL will affect transcriptional regulation ⁃ RESULTS: Diversity of use for SARMs on the body ION CHANNELS AS DRUG TARGETS WHAT ARE ION CHANNELS ◦ Proteins the span cell membranes that form pores that allows flow of ions across plasma membrane & intracellular organelles CLASSES OF ION CHANNELS ◦ Ligand gated: requires binding of protein on the ion channel so that pores of channels can open or close ◦ Voltage gated: channels that open depending on membrane potential ◦ Physical cue: activated by the environment like pressure or temperature ◦ Other ion channels: ⁃ Sodium leak channels: Pores that are always open LIGAND GATED ION CHANNELS ◦ Large class that includes many neurotransmitter receptors ⁃ Found in central & peripheral nervous systems & other channels found on cells throughout the body ◦ Ionotropic glutamate receptors ⁃ Ionotropic: denotes that its an ion channel & distinguishes from metabotropic glutamate receptors ◦ Nicotinic acetylcholine receptors: target for nicotine ⁃ Ion channels and NOT metabotropic: muscarinic acetylcholine receptor ⁃ Diff. B/w nicotinic & muscarinic: ⁃ Nicotinic: channel protein, when acetylcho. binds = causes diffusion of cations  ⁃ Muscarinic: membrane protein, when stimulated by neurotransmitter = opening of ion channel indirectly through 2nd messenger ◦ Inhibitory Neuron Ion channels: ⁃ GABA, glycine, serotonin ◦ Found & beyond CNS: ⁃ On PPT: Acid sensing (proton gated) ion channels (ASICs) & everything after VOLTAGE GATED ION CHANNEL PROTEIN SUPERFAMILY ◦ 143 members across 8 classes (everthing else was read off slide) PORE REGION FAMILY TREE ◦ Cyclic nucleotide channels are in 2 clusters (CNG & HCN) ⁃ B/C thr respond to hyperpolarization ◦ Potassium channels = largest group of voltage gated ion channel TRANSIENT RECEPTOR POTENTIAL CHANNELS (TRPC) ◦ Opens as response to environmental cues ⁃ Tissue acidosis result of damage to cells and that is considered pain in the CNS ◦ TRP channels act as signal transducer by altering cells membrane potential or increasing intracellular calcium conc. ◦ TRPC insensitive to voltage ◦ 6 helices that go across plasma membrane ◦ Functional TRPC are homotetramers ⁃ They can Flux cations (Na, Ca, Mg) ◦ Temp. Sensitive by Channel: ⁃ Cold: TRPM8 & TRPA1 ⁃ Hot: TRPV1 & TRPV2 OTHER CHANNELS ◦ Aquaporins: ⁃ Channel where water can pass through plasma membrane ⁃ Found on cells that have responsibility for moving alot of water ⁃ Water can diffuse through plasma membrane on its own but its slow ⁃ Using aquaporin channel can make water diffuse faster ⁃ Electrostatic interactiins make pore selective for water ⁃ Small subset of aquaporin that can transport glycerol ◦ Stretch activated receptors: ⁃ Includes some TRPCs, and other members like Piezo 1 ⁃ cation channels that mediate membrane depolarization  ⁃ Stretch or deformation that Piezo 1 is responsive to is restricted to the plasma membrane its immediate vicinity ◦ Connexins & Pannexins: ⁃ hemi-channels ⁃ Allows passage of cations, anions, & small molecules ( electrical force so K+ continues to move out cell ⁃ Eq. Potential is reached when conc. gradient = electrical force (last picture) ⁃ K+ stop going out cell b/c the elec. force overcomes the draw of conc. gradient ⁃ Open channel doesn’t matter anymore there is no net movement @ eq. potential ION FLOW, EXAMPLE: NEURONS ◦ Resting membrane potential (hypothetical): - 70mV EQ POTENTIAL(mV) ION CONC. FLOW K+ - 88 In >> out Out Na+ + 60 Out >> in In Cl- - 61 Out >> in Out ◦ Potassium: PREVIOUS SLIDE, conc. Gradient stronger ◦ Sodium: ⁃ Conc. Is higher out than in, so Conc. Gradient want ions to flow in cell ⁃ conc. Gradient & elec. force works to pull sodium ions IN cell ⁃ They work in same direction ⁃ Ensures high movement of Na+ INTO the cell ◦ Chloride: ⁃ conc. Of Cl- is high out the cell than in ⁃ Cl- wants to move in if only obeying conc. Gradient ⁃ BUT b/c neuron is -70 mV (more negative) than the movement of chloride ions into the cell ⁃ SO, since Cl- is negatively chargerd its going to be REPEL by the - 70mV  ⁃ Repelent elec. force is stronger than conc. Gradient ◦ BOTTOM LINE: ⁃ If Eq. Potential is lower than resting membrane potential: ions flow OUT ⁃ If Eq. Potential is higher than resting membrane potential: ions flow IN NEUROTRANMISSION RELIES ON LIGAND GATED & VOLTAGE GATED ION CHANNELS ◦ release of neurotransmitter into synapse & recieve by post synapitc cell ◦ Steps of neurotransmitter ◦ Excitatory postsynapitc potential (EPSP): cations raises membrane potential closer to threshold (depolarization) ◦ When threshold is reached an action potential will fire at the axon hillock POST SYNAPTIC POTENTIALS ◦ Inhibitory Postsynaptic potential (IPSP) ⁃ Happens when ligand gated ion channels are permeable to chlorine ions  ⁃ Lowers membrane potential below resting potential (hyperpolarization) ◦ Action potential firing at the axon hillock depends on sum EPSP & IPSP ⁃ EPSP & IPSP add up to get cell to threshold then action potenial will be fired PHYSIOLOGY OF VOLTAGE GATED ION CHANNELS ◦ NO PHYSIOLOGICAL LIGAND ◦ Voltage sensor made of charged amino acids ⁃ Position is altered with membrane depolarization ⁃ Shift allows passage of ions that can go through channel ◦ In picutre of Na+ channel: ⁃ Movement of cations into the cell will depolarize membrane further amd allow more channels to open ◦ Voltage gated ions after opening have periods of inactiveness and dont allow passage of ions VOLTAGE GATED CHANNEL MODULATION ◦ Many ion channels posses regulatory sites ⁃ They govern whether ion will open or nit with depolarization ◦ Picture shwos ion channel being modulated: ⁃ If 1 PKG & 1 PKA binds to Calcium channel 1.3 = basal condition, ions pass  ⁃ if 2 PKG binds/phosphorylates to Ca1.3 = inhibition of flow of ions ⁃ If 2 PKA binds/phosphorylates to Ca1.3 = more ions can pass, stronger flux ◦ Other modes of ion channel regualtion ⁃ Channels that can be activsted by other ions ⁃ In picture: Ca2+ channel has cations influx/going in cell can shift the membrane potential and induce movement of K+ out of cell PHYSIOLOGY OF LIGAND GATED ION CHANNELS ◦ Opening of pore happens through ligand binding ◦ The movement of ions is driven by electrochemical gradient ◦ Ligands can bind to: ⁃ Primary site AKA orthosteric site ⁃ Allosteric sites - allows altering of conformational change or timing ⁃ Effect is nit sake to direct ligand binding LGIC: PERMEABKE TO Na+, Ca2+, K+, Cl- ◦ Major excitatory LGIC in the body is Glutamate receptor ◦ NMDA receptor opens by co activation of 2 ligands ⁃ Glutamate and glycine needs to bind to receptor of it to open ◦ Negative regulation of NMDA receptor happens by: ⁃ Blocking of pore by magnesium or zinc ◦ Primary receptor @ neuromuscular junction: nicotinic acetylcholine receptor ⁃ Permeable to sodium and potassium ⁃ Its ligands: nicotine & acetylcholine ◦ 2 Intracellular receptors: IP 3 & Ryanodine ◦ Major inhibitory receptors of CNS: GABA & Glycine receptors ⁃ Lowers action potential firing by keeping membrane potential negative ◦ Zinc activated channel ⁃ ligands: protons (H+), copper, zinc ION CHANNELS AS DRUG TARGETS ION CHANNEL MODUALTORS: ADDICTION ◦ Mesolimbic system: brains reward system ◦ Nicotine increases firing of dopaminergic neuron ⁃ Hapepns by allowing sodium flux, thus bringing cells closer to firing threshold ◦ Chantix is a competitive partial agonist ⁃ It competes with nicotine for binding to the nicotinic acetylcholine receptor ⁃ But Chantix doesnt have to open the receptor as effectively as nicotine ⁃ Ultimately reducing neuron activity while delivering some of the reward ION CHANNEL MODUALTORS ◦ Positive allosteric modulator of GABA A receptor by: alter channel structure Reducing action potential firing, increase Cl- flux, ◦ Tolerance: initial dose is not capable of eliciting the same physiological response = Thus patient needs higher dose to achieve that effect ION CHANNEL MODULATORS: BLOOD PRESSURE ◦ Calcium channels blockers are LEAD class of ion channel inhibitors used to treat heart disease ⁃ Calcium is a trigger for heart muscle contraction ⁃ By regulating calcium = lowers or increase heart muscle contraction ◦ Amlodipine is an ANTOGONIST for Ca1.3 channel ION CHANNEL MODULATORS: ELECTROLYTE BALANCE ◦ read from slides ION CHANNEL MODULATORS: GLYCEMIC CONTROL ◦ Family of compunds used to trest type 2 diabetes PHYSIOLOGY OF INSULIN SECRETION 1. glucose transported into beta cell 2. ATP that is made with that glucose reduces activity of K+ channel 3. Reduced activity of K+ channel = depolarizing cell and Na+ influx 4. Sodium further depolarizes cells to allows Ca2+ influx 5. Insulins released ◦ Anither way: Nateglinide & repaglinide can block potassium channel so you dont need to have glucose, you can just start from STEP 3 TARGETING EVENT DOWNSTREAM OF ION CHANNELS ◦ Gabapentin ⁃ Was thought to inhibit voltage gated calcium channels ⁃ Actually: indirect reduction of ion channel conductance through limiting the traffic of a ligand gated ion channel, NMDA receptor to plasma membrane ⁃ Works by reducing # of NMDA complexes @ synapse ◦ Activation of P2X receptors, triggering AMPA receptor internalization by Ca2+ influx ⁃ Significantly modualte glutamate signaling @ synapse ION CHANNEL DRUG DISCOVERY ◦ ~15% drugs target ion channels KINASES AS DRUG TARGETS KINASE FUNCTION ◦ Protien kinases: enzymes that phosphorylate proteins on tyrosine, serine, threonine residues ⁃ Phosphorylation: installation of phosphate group on alcohol ⁃ Phosphate group comes from ATP ◦ Kinases generally phosphorylate tyrosine OR serine and threonine ⁃ Structure similarity b/w serine and threonine (only differs by 1 methyl group) ◦ In image of simplified protein of tryrosine, serine, threonine ⁃ serine/threonine kinases can phosphorylate either serine or threonine ⁃ Tyrosine kinases can phosphorylate only at tyrosine RECEPTOR KINASES ◦ Cytoplasmic kinases (inside cell @ cytoplasm - intracellular) ◦ Receptor Kinases (transmembrane - in and out the cell) ⁃ ATP binding site located on intracellular/inside domain ◦ Small kinase inhibitors bind @ intracellular domain ◦ Ligand binding @ extracellular domain ⁃ Extracellular growth factors also bind to extracellular domain ◦ Biological therapeutics (monoclonal antibodies) that target kinases targets @ extracellular domain ◦ Kinase dimer: two kinases exists @ cell surface ⁃ Activated by growth factors ⁃ Receptor does autophosphorylation(image showing transphosphorylation) ⁃ Converting ATP to ADP and then using the phosphate to phosphorylate itself ⁃ Transphosphorylation: one monomer of dimer phosphorylates the partner monomer (same on the other side) ⁃ After phosphorylation, it attracts intracellular protein ⁃ Many times intracellular protein are chtoplasmic kinases ⁃ Intiates downstream cell signalling process KINASE INHIBITOR DRUG HISTORY ◦ Read from slide KINASES AND CANCER ◦ Relationship b/w cancer and kinases: ⁃ Abnormal kinases contributes to: ⁃ survival: makes cancer cell resistant to apoptosis ⁃ motility: ability of cell to move in body, leads to metastasis  ⁃ evading immunity: prevent immune system from attacking cancer cells ⁃ proliferation: uncontrolled growth of cancer ⁃ angiogenesis: formation of new blood vessels to feed cancer ⁃ metabolism: increased uptake of glucose leadig to proliferatation GENETIC MUTATION AND KINASES ◦ Single point mutations: change to single nucleotide resulting in wrong AA ⁃ Leads to kinase being active all the time (constitutively active) ◦ Gene amplification: multiple copies of gene ⁃ Leads to overexpression of kinase (not mutated structually but too many copies) ◦ Chromosome rearrangement: sequence form single chromosome becomes disordered, sometimes parts of 2 chromosomes combine to form new chromosome and then new protien ⁃ Sometimes gene encoding kinsase ends next to a strong promoter gene thus over expression can happen KINASES AND SIGNALING PATHWAYS ◦ Cell signaling: informstion transferred from cell surface>cytosol>nucleus leading to changes in gene expression ⁃ Starts w/ extracellular signaling molecules and transmembrane proteins  ⁃ Ends w/ Intracellular domain results in cascade of info that goes into nucleus where transcription & gene expression takes place ◦ Drugs target a multiple sites of the kinase signaling pathway ⁃ B/c stopping signialling @ point of pathway is not enough ⁃ Some cancers can develop resistance to kinase inhibitors thus inhibiton @ multiple sites can stop the pathway for good ◦ In the image above: MAPK is left pathway, PI3K is right pathway BIOLOGIC AS KINASE INHIBITORS ◦ monoclonal antibodies are large molecules that recognize specific csmcer associated proteins and bind to antiges @ CELL SURFACE (extracellular) ◦ Since receptor kinases have a extracellular domain monoclonal antibodies can bind to those ⁃ thus making receptor kinases targets for monoclonal antibody therapy ◦ Kinase inhibition my MAbs by multiple mechanism: ⁃ Block binding of growth factor or hormone responsible for kinase activating ⁃ Prevent change in conformation necessary for intracellualr domain activation ⁃ Prevent dimerization @ cell surface (stopping the 1st step in functionality of receptor kinases) ◦ Monoclonal anitbody drugs ALWAYS ends in “mab” SMALL MOLECULE KINASE INHIBITORS ◦ Type 1: DFG is inward ◦ Type 2: DFG outward ⁃ Type 1 & 2 are competive ATP inhibitors , most drug are ATP competitive ◦ Type 5 uses a chemical linker to join ATP site and distal site ◦ Type 6: forms irreversible/covalent bonds b/w protein & drug ⁃ Advantage of irreversible bonds: ⁃ Once kinase is modified by covalent drug it’s permanently inactivated ⁃ Only way to get it active again is if new proteins are synthesized ⁃ Its importamt for covalent bond to be controlled only the intended kinase should be modified by covalent inhibitor or else there will be target toxicity TYPE 1 VS TYPE 2 EXAMPLE ◦ “ib” = used for all small molecules drug inhibitors ◦ Imatinib (pink): Type 2 ⁃ Opens additional binidng pocket within kinase ATP binding site b/c the Phe382 is facing outward ⁃ Occupied by amide protion of imatinib ◦ Dasatinib (green): Type 1 ⁃ Phen382 closes off back pocket thus imatinib cannot bind in this area ◦ Resistance to imatinib is caused by point mutations that stabilize ezyme in active confirmation ⁃ Mutant kinases seems to preset in its ONLY active form thus imatinib cant bind ◦ Dasatinib inhibits all imatinib resistant mutation EXCEPT FOR: ⁃ Threonine to isoleucine mutation @ residue 315 ⁃ Its a gatekeeper residue for BCRABL ⁃ AKA gatekeeper mutation ATP BINDING SITE INHIBITORS ◦ hinge region /beta strand: ⁃ ATP comp. Inhibitors almost always make H-Bonding interactions w/ hinge region ◦ Gatekeeper Residue mutation that increase the size of residue often leads to drug resistance ◦ 2 hydrophobic region on either side of the adenine in ATP ⁃ Hydrophobic fucn. Groups on kinases ocupie either one or both areas ◦ Solvent accessible: often install polar/water solublizing groups/water solubility enhancing groups on inhibitor molecule to increase solubility GEFITINIB EXAMPLE ◦ TYPE 1 ◦ Used to treat non small cell lung cancer (NSCLC) ◦ Activating mutation happen in EFGR to drive proliferation TYPE VI(6) COVALENT KINASE INHIBITORS ◦ Chemical warheads: attached to drug & react w/ AA residue @ ATP binding site (remeber this is irreversible covalent bonding) ⁃ AA residue like: cysteine, lysine, aspartic acid ◦ Survey showed: front pocket & extended front pocket are mkst common location for targeted residues ◦ Use of alpha beta amide targeting cystine in front pocket of ATP binding site = most successful ALPHA BETA UNSATURATED AMIDE WARHEAD ◦ Image:  ⁃ Cystine residue in front pocket of kinase ⁃ Thiol on cystine is nucleophilic ⁃ Nucleophile react w/ ab unsaturated amide (electrophilic) ⁃ Mechanism: nucelophile (thiol) attacks beta carbon on electrophile (amide) ⁃ End result: formation of covalent bond ASSAY IN KINASE DRUG DISCOVERY ◦ Binding assays: affinity of ligand for specific binding site on kinase ⁃ Ligand under investigation(experimental drug) competes for binding w/ known ligand and then the displacement can be measured ◦ Biochemical Kinase Assays (enzymatic assays): not cell based assay, measures ability of enzyme to function (consumption of substrate vs production of product) ◦ Cell based phosphorylation assay: measuring phosphorylation event that downstream of kinase of interest (most frequently used, and good way to assessmfunction of kinase) ◦ Cell proliferation assays: meausre growth of tranformed cells in culture and the ability of kinase to stop the growth ⁃ Example of phenotypic assay (doenst tell about mechanism) GENERAL KINASE INHIBITOR PROPERTIES ◦ More than 1/2 small molecuke kinase inhibitor ps (SMKIs) drugs have MW of 450 and up ◦ Drugs are lipophilic, hughky bound to plasma proteins ◦ Most SMKIs are dosed orally (tablets or capsules) one is dosed via IV ◦ SMKIs metabolized by cytochrome P450 enzymes w/ CYP3A4 playing vital role in metabolism (located in liver)