Drug Discovery and Design A Proteins: Fundamentals

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Ferdinand Pearson
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Drug Discvery and Design A Prteins: Fundamentals Prteins are heter-plymers (plypeptides) f cvalently linked a-amin acids as well as the additin f c-factrs (metal ins), cenzymes, prsthetic grups r

1 Drug Discvery and Design A Prteins: Fundamentals Prteins are heter-plymers (plypeptides) f cvalently linked a-amin acids as well as the additin f c-factrs (metal ins), cenzymes, prsthetic grups r ther mdificatins. Cfactr: general functinal nn-amin acid cmpnent metal ins, rganic mlecules Cenzyme: used t designate an rganic c-factr NAD + in lactate dehydrgenase Prsthetic grups: cvalently attached c-factrs heme in myglbin Prteins have varied functins: In catalysis: such as enlase in glyclytic pathway, DNA plymerase in DNA replicatin In transprt: haemglbin transprts O 2, lactse permease transprts lactase acrss cell membranes In structure: such as cllagen cnnective tissue r keratin in nails, hair, feathers and hrns. In mtin: including mysin (muscle tissue) and actin (muscle tissue and cell mtility) Imprtant t knw where drug/ligand binds t a prtein and why binding ccurs at ne prtein but nt anther (differences). Als imprtant t knw why a prtein is stable r why it aggregates r why it is sluble/insluble. Prtein structure: Primary structure: the linear sequence f amin acids/amin acid residues. Each amin acid has a unique structure (due t uniqueness f side chain). Structure and chemistry is available in building functinality. Secndary structure: certain sequences frm H bnds that cause the regin t fld int a spiral (a helices), a sheet (b sheets/strands) and lps/cils. b sheets must have tw strands. Ramachandran plt: phi and psi angles frm cperative patterns called the secndary structure and are relatively free t rtate. The plt visualises energetically allwed regins fr backbne dihedral angles phi and psi f amin acids residues in prtein structure Intrns and exns: eukarytic genes are cmpsed f exns (prtein cding sequences) and intrns (nncding sequences). Intrns remved after primary transcriptin by splicing. RNA is stabilised by 7-methyl guansine (5 cap) and ply A tail. Alternate splicing: using same gene t create different prteins exns can be jined tgether in different cmbinatins and hence different prteins are frmed with varied functins. Pst translatinal mdificatin (prtelytic prcessing) pre-sequence is the signal peptide, the prsequence is t be remved t generate the mature prtein Tertiary structure: the arrangement f secndary structure elements. Quaternary structure: mlecules f prteins assciate t frm multimers. Bnds include: cvalent (backbne and disulphide bridges), nn-cvalent (VDW), hydrgen bnds and salt bridges. Structure and naming f amin acids General structure f an amin acid: Amin acids have prperties that are well suited t carry ut a variety f bilgical functins because f their capacity t plymerise, useful acid-base prperties, varied physical prperties and varied chemical functinality. Peptide bnds are planar; planarity is assciated with delcalised electrns (partial duble bnd) Amin acids in 5 classes: Nn-plar, aliphatic (hydrphbic): glycine, alanine, prline, valine, leucine and methinine. Armatic grups absrb UV light: phenylalanine, tyrsine and tryptphan. Plar (hydrphilic) frm H/S-S bnds: serine, threnine, cysteine, asparagine and glutamine Psitively charged (basic): lysine, arginine and histidine Negatively charged (acidic): aspartate and glutamate (aspartic acid and glutamic acid) Zahra AR 1

2 Uncmmn a.as in prteins are nt incrprated by ribsmes. They arise by pst-translatinal reversible mdificatins f prteins especially phsphrylatin (imprtant in regulatin and signalling) Inisatin behaviur f amin acids and peptides At acidic ph the carbxyl grup is prtnated and the amin acid is in the catinic frm At neutral ph the carbxy grup is deprtnated but amin grup is prtnated. The net charge is zer and the in is a zwitterin. At alkaline ph the amin grup is neutral, the carbxylic acid is in the aninic frm. The chemical envirnment affects pka values a-carbxyl grup much mre acidic than in carbxylic acids, a-amin grup is slightly less basic than in amines. Perturbatins in pka may ccur frm nearby interactins. Amin acids may act is buffers. Amin acids with uncharged side chains (e.g. glycine) have tw pka values and hence can act as buffers in tw ph regimes. Zwitterins predminate at ph values between the pka values f the amin acid and carbxyl grup. The iselectric pint (pi) f an amin acid is the pint in which the net charge is 0, the amin acids are least sluble in water and will nt migrate in an electrical field. Fr amin acids withut inisable side chains: pi = pka 1 + pka 2 / 2 Inisable side chains can als be titrated but titratin curves becme mre cmplex. pk a values are discernible n the curve if they are mre than tw ph units apart. Fr amin acids with inisable side chains t find pi: Identify the species that carries a net 0 charge Identify the pka value that defines the acid strength f this zwitterin Identify the pka value that defines the base strength f the zwitterin Take the average f the tw values (average f pk a and pk R ) All amin acids (except glycine) are chiral. The a carbn has 4 different substituents and is tetrahedral each amin acid has a unique substituent (R grup), in glycine the R grup is a hydrgen. All amin acids (except prline) have an acidic carbxyl grup, a basic amin grup and an a hydrgen cnnected t the a carbn. Stereismers are nn-superimpsable chemical ismers that have identical cvalent structure and exist fr all chiral amin acids. There are tw classes f stereismers: enantimers & diasteremers. Enantimers have either an L r D rtatin CORN rule clckwise COOH à R à NH is L and anticlckwise is D L-alanine is the naturally ccurring frm f alanine Structure and prperties f peptides Small cndensatin prducts f amin acids (mw < 10kDa) Peptides have a variety f functins that include: Hrmnes and phermnes: e.g. insulin, xytcin, sex peptide. Neurpeptides: e.g. substance P (pain mediatr) Antibitics: e.g. plymyxin B fr gram negative and bacitracin fr gram psitive Prtectin: txins e.g. amanitin (mushrms), cntxin (cne snails), chlrtxin (scrpins) Zahra AR 2

3 Fates f Dietary Carbhydrates Nearly all dietary carbhydrates are starch (plymer f glucse). There are tw frms f starch: Amylse: linear, frms helices, difficult t digest Amylpectin: branched, easy t digest, pst-prandial respnse like pure glucse Disaccharides: Lactse: galactse + glucse, cnsequences f lactase deficiency (lactse intlerance) Sucrse: fructse + glucse Maltse: glucse + glucse Mnsaccharides: Glucse Fructse: fruit and hney Insulin: Synthesised in the endcrine part f the pancreas by b-cells f the islets f Langerhans When glucse is ingested, bld glucse rises and this stimulates the pancreas t release insulin. There are als ther stimulants. Insulin causes bld glucse t fall because it causes glucse t be taken up by the tissues. It als supresses the liver s ability t make glucse Glucse respnses: aim t maintain bld glucse cncentratin at ~5mM. Levels less than 3mM wuld lead t cma. Prlnged high glucse cncentratin may lead t glycsylatin f prteins. Cnsequences f intlerance: Pst-prandial hyperglycaemia: if it ccurs after every meal and persists fr several hurs then there will be prblems. The persn will rarely be eugylcemic, and may lead t cmplicatins f hyperglycaemia and prtein glycsylatin. Rt cause may be insulin resistance: impaired ability t respnd t insulin (underlies type 2 diabetes) Cntrl f glucse intlerance: cnsumptin f slwly absrbed starches The glycemic index: describes pst-prandial glucse respnse (area under the test fd glucse curve divided by the area under a reference fd glucse curve). Reference fd is nrmally 50g glucse and the test fd is given in an amunt that will give the same amunt f digestible carbhydrate. Rati is expressed as a percentage: GI f mdern, prcessed amylpectin fds is > 80%, GI f ld wrld grains and legumes is < 30%. By definitin, GI f glucse wuld be 100%. The GI is useful knwledge fr cntrlling bld glucse especially in relevance t diabetes ( BGL). Quality f a carbhydrate (its GI) is as imprtant as the ttal amunt f the carbhydrate. Sugary (sucrse) fds have a lw GI because half the carbhydrate is fructse. Fructse cntaining fds are lw GI. Dairy fds are lw GI because half the carbhydrate is galactse (prtein elicits insulin secretin). GI is assessed as respnse t 50g f absrbable test CHO (nt all f which is glucse). Glucse uptake in muscle and white adipse tissue: GLUT-1 and GLUT-4 bring the glucse int the cells. GLUT-1 is nt under insulin cntrl and GLUT-4 is. GLUT-4 is present in the cell as vesicles f Glgi and under the influence f insulin, it translcates t the t the cell surface t facilitate the entry f glucse. Zahra AR 3

4 Inside the cell, hexkinase phsphrylates the glucse (G6-P). This requires ATP. The glucse 6-phsphate may then enter the glyclysis pathway when energy is needed via the rate limiting enzyme phsphfructkinase If energy is nt needed, the glucse will be used in glycgenesis (making glycgen) facilitated by the glycgen synthase enzyme If glucse 6-P builds up, it will inhibit the hexkinase s that phsphrylatin stps and glucse can easily exit the cell Hexse metablism glycgen frmatin: glucse is phsphrylated (G 6-P), then rearranged t frm glucse 1-phsphate. Then using UTP (releases PP) frms a UDP glucse (activated glucse). PP hydrlysis pulls reactin t cmpletin. The UDP glucse then adds n t the glycgen chain via glycgen synthase. Glycgen is a plymer f glucse with glucse residues. Bdy cannt stre a lt f glycgen because it is branched and hence a heavy mlecule. UDP needs t be regenerated t UTP = UDP + ATP à UTP + ADP (cst 1 ATP t add ne glucse t the glycgen chain) Glycgen synthase: Glycgen synthase with a phsphate attached is inactive. It is regulated by reversible phsphrylatin (cvalent mdificatin) a phsphate grup is remved via the enzyme prtein phsphatase I (PPI). Insulin stimulates this (stimulates glycgen synthesis) T deactivate the activated enzyme, it is phsphrylated back in a prcess catalysed by glycgen synthase kinase. Phsphfructkinase: catalyses the secnd energy investment stage f glyclysis. It is regulated allsterically stimulated by lw energy charge (when high ADP, AMP and lw ATP). Small change in ATP, ADP causes a large relative change in AMP. AMP mlecules bind at a site away frm the active site (allsteric binding site) Cupling: stimulatin f glycgen synthase by insulin creates an energy demand. Glycgenesis is anablic s trapping and activatin f glucse requires ATP. Drp in energy charge stimulates PFK hence insulin has indirectly stimulated PFK and glucse xidatin Anablic pathway requires catablic pathway i.e. signal t stre fuel als causes fuel burning Liver glucse uptake: GLUT-2 is used t take up glucse frm bld in the liver and pancreas very high activity and very abundant ([glucse] in bld = [glucse] in liver) Gluckinase (variant f hexkinase) rapidly cnvert G à G6P. it is nt inhibited by build-up f prduct. High K M (10mM) fr glucse nt saturated by high levels f liver glucse s [G6P] rapidly increases as [glucse] in bld rises G6P in liver can stimulate inactive glycgen synthase in liver even phsphrylated GS Glycgenesis: In liver: the push mechanism [G6P] can get high enugh t stimulate GS Glycgenesis respnds t bld glucse withut the need f insulin Althugh insulin will stimulate glycgenesis further In muscle: the pull mechanism [G6P] never gets high enugh t stimulate GS insulin stimulates GS and pulls glucse int glycgen In bth liver and muscle: 2 ATPs required fr the incrpratin f a glucse int glycgen chain branching enzyme needed t intrduce a1à6 branch pints transfers a segment frm ne chain t anther limit the size f glycgen mlecule branches becme t crwded even if they becme prgressively shrter Lipgenesis: prcess f cnverting acetyl CA back int fatty acid. It is stimulated when insulin binds t its receptr n the cell surface and inhibited by dietary fat (high intake). The prcess cnsumes ATP and a reductant (prduced in glyclysis). NADH released in the Krebs cycle ultimately prduces the ATP that is Zahra AR 4

5 cnsumed in lipgenesis. Fllwing lipgenesis, three fatty acids jin t a glycerl t frm a triglyceride in an energy requiring prcess called esterificatin. Pyruvate dehydrgenase: cnverts pyruvate t acetyl CA in the mitchndria. Insulin stimulates PDH phsphatase. Cenzyme A alne can t crss membranes. Fate f acetyl CA: depends n the energy need (charge) and stimulus driving lipgenesis (e.g. insulin). Catablic: xidatin, when there is little energy in the mitchndria it is burnt in the Krebs cycle. Carbn is fully xidised t CO 2 and NADH is prduced t generate ATP. Anablic: transprt, when there is a lt f energy it ges t lipgenesis. Carbns are mved t the cytplasm and activated fr fatty acid synthesis. First step in bth is citrate frmatin because the big CA grup can t leave the mitchndria ATP-citrate lyase (ACL): Once in the cytplasm, citrate is cleaved using CA t generate acetyl CA and xalacetate Reactin requires cnversin f ATP à ADP+Pi and is catalysed by ATP citrate lyase (ACL) ACL is inhibited by hydrxyl-citrate (OHCit) Acetyl CA carbxylase (ACC): activates acetyl CA and primes it fr lipgenesis. It is unusual in that it fixes carbn dixide in the frm f bicarbnate (carbxylatin): acetyl CA + CO 2 à malnyl CA Carbxylatin reactin requires cnversin f ATP t ADP and Pi It is the rate limiting step fr lipgenesis. Requires bitin as a c-factr. Stimulated by insulin malnyl CA is cmmitted t lipgenesis à reversible phsphrylatin Regulated allsterically stimulated by citrate (plymerisatin f the enzyme) and inhibited by lng-chain fatty acyl CA. (eventual prduct f lipgenesis) Fatty acyl synthase: makes fatty acids. Requires reductant (NADPH). Gene expressin is stimulated by insulin à mrna. There are tw free SH grups n an acyl-carrying prtein which keeps the intermediates in the right psitin fr interactin with the right active sites each new 2C unit is added nt the carbxy end. Additin sequence: each rund f 2 carbn additin requires 2 NADPH but n ATP. Release f CO 2 that went n during the prductin f malnyl CA (carbxylatin f acetyl CA desn t fix CO 2 ). Fatty acids then get released as FA-CA when chain length is greater than ~ 14 carbns. Desaturatin (adding duble bnds) is dne after FA synthase. Desaturated fatty acids: duble bnds are in cis (H n same side) frm. Trans fats in fds can be harmful because they can t be prcessed by enzymes Essential fatty acids: cannt be synthesised by humans because the bdy lacks the required desaturase enzymes, hence are essential in a diet. Include mega 3 and 6 fatty acids. Pentse phsphate pathway: prvides reductant (NADPH) fr lipgenesis: NADPH frm f NADH invlved in the anablic reactins NADH prductin by PPP is prprtinal t demand Early reactins xidise and rearrange G6P, generating NADPH and 5C sugar phsphate Later reactins xidise and rearrange carbns t allw re-entry t glyclysis PPP is als used t make ribse-5-phsphate fr DNA, RNA, NAD, CA and ATP. NADPH als acts as an antixidant red bld cell deficiency in first enzyme in PPP can cause anaemia Esterificatin: frmatin f fat (glycerl + 3 fatty acids). Glycerl needs t be in the glycerl 3-phsphate frm frm reductin f glyceraldehyde 3-phsphate fr glyclysis. Glyclysis is imprtant fr prductin f bth acetyl CA and glycerl. Esterificatin enzymes use FA-CA fatty acids added ne at a time Bth esterificatin enzyme and FAS are unregulated by insulin, regulated by insulin stimulated gene expressin and prtein synthesis. Fatty acids dwn regulate when there is an abundance f fat Zahra AR 5

6 DNA Repair Repair f DNA is vital t the survival f species. Unlike RNA, DNA has the capability t repair itself. The extra cpy prvides the template and elabrate repair mechanisms have evlved t crrect crruptins. Many errrs are crrected by the 3 à 5 exnuclease activity f DNA pls I and III. There are crruptins t the sequence which ccur after replicatin. 130 genes which encde prteins are respnsible fr repair in the human genme. When DNA is expsed t UV radiatin, it frms pyrimidine dimers. C5 and C6 bnds f tw cnsecutive pyrimidines frm cvalent bnds with each ther (cyclbutyl ring). Cyclbutyl bnds are ~1.6 Å s are much shrter than distance between bases (~3.4Å). This causes a bulge in the DNA duble helix which prevents transcriptin and replicatin. There are ways t repair the dimer. One way is t cleave the bnds directly with phtlyase. This enzyme uses the energy frm visible light t cleave the cyclbutyl bnd. Phtlyase is present in many prkarytes and eukarytes but nt in humans. Anther type f repair is nucletide excisin repair. The pyrimidine dimer and adjining nucletides are cut ut by excisin enzymes. The enzymes seek ut the bulge and cut it ut. DNA pl I then fills ut the missing sectins using the secnd strand as a template. The gap is sealed by DNA ligase. Uracil is created by spntaneus deaminatin f cytsine, and des nt belng in the DNA. A set f excisin enzymes (base excisin repeair) will cleave the base at the glycsidic bnd (depurinatin) leaving an apurinic r apyrimidinic site. Dexyribse + adjining nucletides are then excised by an AP endnuclease. The whle sectin is filled with DNA pl I and sealed with ligase. A number f anti-cancer drugs that induce DNA damage and apptsis (e.g. Dxrubicin). Anti-cancer drugs used t treat brain tumrs interfere with replicatin. Ability f cells t repair this damage limits the effectiveness f the drug. Success f these therapies will depend n the activity f repair enzymes. Mutatins: a mutant gene has a different sequence t the wild type gene. The change is inheritable, hwever the mutatin may r may nt cause a change in phentype. Fr a mutatin t be inherited, it must be present in the germ line cells. Mutatins in smatic cells r in the RNA cde will nt be inherited. Static mutatins: change in the cde becmes a stable incrpratin int the genme f the germline cells as well as all smatic cells in the rganism. The change is transferred t the next generatin. Examples include PKU, cystic fibrsis and sickle cell anaemia. Expressin in the phentype depends n genetic infrmatin frm bth parents and epigenetic factrs. Dynamic mutatins: mutatins increase in severity with each generatin, varies between tissues f the same rganism. Examples include Trinucletide repeats (TNR). These mutatins lead t genetic anticipatin. The fllwing generatin will be mre affected and/r have an earlier nset. Can ccur whenever DNA is being cpied Frmatin f aberrant lp structure when DNA strands are separated Types f mutatins: Transversin: purine replaced by pyrimidine r vice versa, implicatins fr 3D shape f helix Transitin: pyrimidine fr pyrimidine r purine fr purine Silent mutatins: altered cdn still cdes fr the same amin acid (cause cde redundancy) Frameshifts: shifts reading frame by adding/deleting bases, leading t nn-functinal prtein Neutral mutatin: altered cdn cdes fr a functinally similar amin acid hence has n effect n the functinality f the prtein Pint mutatins: single base pair change, can be a substitutin, deletin r additin Missense: altered cdn fr functinally different amin acid. Can be lethal. Nnsense mutatin: prduces a stp cdn and causes a truncated prtein. Very dangerus. Splice mutatin: prduces r remves a splice site nly in eukarytes Temperature sensitive: mutatin causes changes in functin f temperature sensitive prtein Prtein functins nrmally at lwer temperature but is inactive at high temperatures. Reversin: a mutatin which reverts t wild type, referred t as revertant Leaky mutatin: desn t affect rganisms in nrmal cnditins nly in stressed cnditins. Mutagen: physical r chemical agent that causes mutatin t ccur at a higher rate r frequency. Zahra AR 6

7 Natural/spntaneus mutagenesis: Occurs at a nrmal backgrund rate all the time. Alternative tautmer f the base if base changes during replicatin, the wrng nucletide will attach and by tw runds f replicatin there will be a base pair switch. An errr in replicatin that is nt picked up by prf-reading activity f DNA pl III will result in a mutatin. Deaminatin f cytsine r adenine t hypxanthine. If the adenine flips t the alternative tautmer at the time f replicatin, just as the new incming nucletide is selected, it will base pair t cytsine rather than thymine as the H dnr and acceptr are the ther way arund. Within anther generatin, there will be a GC instead f an AT These can be crrected by specific repair mechanisms Induced mutagenesis: Intercalatrs: planar, ring structures with slide in between the pairs causing a disruptin t nrmal base stacking (e.g. ethidium brmide) Alkylating agents: methylate r ethylate bases result in altered base pairing during replicatin Base altering agents: e.g. nitrus acid cnverts amin (dnrs) t ket (acceptrs) grups Base analgues: e.g. 5-brmuracil replaces thymine but base pairs t guanine Testing mutagenesis: Ames test. Quick screening test fr ptential mutagenic cmpund Strain f salmnella with defect in histidine bisynthetic pathway is plated ut n a medium cntaining minimal histidine (enugh t keep cells alive, but nt fr prliferatin) Lking fr a reversin mutatin as bacteria is already a mutant Cmpund f interest applied t a disk in the centre f a plate and incubated ver night Different plates with increasing amunts f the cmpund are put up If cmpund is mutagenic, it will cause a number f cells t revert t wild type and grw n the medium. Linear dse-respnse curve. The mre clnies frming arund the disk, the mre mutagenic it is Nn-mutagenic cmpunds will have a few clnies scattered ver the whle plate Zahra AR 7

Module 3. Chapter 5 Microbial Metabolism. Catabolic and Anabolic Reactions Metabolism Two general types of metabolic reactions: o :

Module 3. Chapter 5 Microbial Metabolism. Catabolic and Anabolic Reactions Metabolism Two general types of metabolic reactions: o : Mdule 3 Chapter 5 Micrbial Metablism Catablic and Anablic Reactins Metablism Tw general types f metablic reactins: : : Recall frm Chapter 2: Energy can be when bnds Energy can be when bnds Catablism Purpse