Nuclear Receptors as Drug Targets - Pharmacodynamics PDF
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University of North Texas Health Science Center
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This document provides an overview of nuclear receptors and their roles as drug targets. The text outlines concepts in pharmacodynamics, including receptor types, ligand interactions, and signaling mechanisms. It also touches upon selective receptor modulators and hormone receptors.
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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)