Peptides: Química de Síntese e Aplicação em Bioquímica, Resumos de Engenharia Florestal

Baixe Peptides: Química de Síntese e Aplicação em Bioquímica e outras Resumos em PDF para Engenharia Florestal, somente na Docsity! Peptídeos: síntese química e seu emprego na pesquisa bioquímica Dr. Robson Lopes de Melo Instituto Butantan Centro de Toxinologia Aplicada CAT is devoted to multidisciplinary research on animal and microbial toxins, to contribute to public science literacy and to transfer the findings stemming from the Center to the COINFAR for drug development CAT - Objectives Avanços em Engenharia de Proteínas e Peptídeos Methods in Peptide Synthesis Formation of a peptide • Enzymatic • Recombinant DNA • Solution synthesis • Solid phase peptide synthesis Avanços em Engenharia de Proteínas e Peptídeos History Emil Fisher Bruce Merrifield 1899-1908 1963 Protection of alpha-amino group Bergman - Zervas (1932) CH2 OH Cl Cl O CH2 O Cl O NH2-CH(R)-COONa CH2 O NH-CH(R)-COONa O NaCl+ + CH2 O NH-R O CH3 CO2 NH2-R H2/Pd Na/NH3 liq. Na2CO3CH2 CH2 CH3 NH2-R HBr/Acetic acid CH2 Br CO2 HBr.NH2-R + + + + + + + t-butyloxycalbonyl group Mckay – Albertson (1957) O NH-R O CO2 NH2-R CH2 CH3 CH3 HCl + Cl - + + HCl+ Avanços em Engenharia de Proteínas e Peptídeos 9-fluorenylmethoxycalbonyl group Carpino (1970) Ai — DAÃo — Go S HO2C FMOCHN Benzyl (Bn) S HO2C FMOCHN tBu t-butyl (tBu) removed with acid removed with acid Ph S HO2C FMOCHN CPh3 trityl (Tr) removed with acid S HO2C FMOCHN NO2 S HO2C FMOCHN S NO2 S HO2C FMOCHN H N O o-nitrobenzyl removed photochemically removed with Ph3Por with HOCH2CH2SH stable to acid removed with HO- or Hg(II) Cysteine Protection of side-chain functional groups Protection of side-chain functional groups Histidine H2N CH C CH2 O O N NH Cl HN CH C CH2 O O N N CH3 -OH CH3 HN CH C CH2 OH O N N H2N CH C CH2 OH O N N HCl/EtOH O HO2C FMOCHN Benzyl (Bn) O HO2C FMOCHN tBu t-butyl (tBu) removed with acid or hydrogenolysis removed with acid Ph O HO2C FMOCHN acetate (Ac) removed with acid O O HO2C FMOCHN CPh3 trityl (Tr) removed with acid Protection of side-chain functional groups Serine, Threonine and Tyrosine H N HO2C FMOCHN 3 NH2 NH2 HONO2, H2SO4 H N HO2C FMOCHN 3 NH2 N NO2 removed with hydrogenolysis or with Zn(0) in acetic acid H N HO2C FMOCHN 3 NH2 N Ts removed with HF Arginine Avanços em Engenharia de Proteínas e Peptídeos Mixed Anhydrides o O o o» A BocNH 7 MN —— >» BocNH o du ——+ EN: 'O'Bu ci R R » 1 o qo —H cent om — vom Am R Ç À AANH NH,AA [6] — cost ut CO, + HO'Bu R Avanços em Engenharia de Proteínas e Peptídeos Active Esters o o BocNH. N O OR R mixed carbonates R='Bu, Bu, cl ci ci o, o BocNH N o R O N-hydroxysuccinimide (HOSu) o BocNH OAr R electron poor aryl esters Ar= pNO,CgHg, CoCls, Cos 1-hydroxybenzotriazole (HOBt) Avanços em Engenharia de Proteínas e Peptídeos carbodiimides NE=CE=EN + polymerization styrene divinylbenzene (crosslinker, ~1 %) initiator Ph Ph Ph Ph PhPhPh Ph Ph Ph Ph Ph Ph Ph H3COCH2Cl ZnCl2 CH2Cl Solid Phase Peptide Synthesis Merrifield Resin Amide-linked resins CH2Cl N - O O 1) 2) H2NNH2 CH2NH2 HN O R1 NHCBz Amide Linked Commercially available K+ Ester-linked Resins CH2Cl O R1 O NHCBz_ O O R1 NHCBz CF3CO2H O O R1 NH2 Commercially available K+ O2N Cl O AlCl3 O NO2 H2NOH•HCl - H2O N NO2 OH N NO2 O O R1 NH2 Kaiser (oxime) resin Wang Resin CH2Cl O OH O O O R1 NH2 Avanços em Engenharia de Proteínas e Peptídeos 4 O 4 O emo eCeom'o % Activation SB Deblocking $ Q Repeat steps for each E amino acid addition BA CNH o h e Final deblock 4 o Dá o po O Q Cleavage and S deprotection o 9 AE Son Free peptide (Final Product) O= EEB coco R CH O C OHNHN- - proteçãoα grupo protetor lateral 2 R CH O C OYNH2 1 H H O R CH O C OYNH R CH O CNHN- - proteçãoα 1 grupo protetor lateral n ( )n R CH O C OYNH R CH O CNH2 1 grupo protetor lateral n ( )n desproteção R CH O C OHNHN- - proteçãoα grupo protetor lateral n N acoplamentos R CH O C OYNH R CH O CNH2 1n ( )n Peptídeo final Solution Peptide Synthesis Microwave Synthesis Solid Phase Peptide Synthesizer •Each step can be performed 10 to 50 times faster with microwave energy •Coupling times as low as 60 sec. have been obtained •Enhanced peptide-resin cleavage in 10 minutes •Microwave reduces amount of excess reagents needed for couplings, which lowers volume of reaction solution •Microwave energy has been shown to decrease racemization •Typical microwave-driven cycles are much shorter, thus racemization is reduced Promotes adrenal steroid production Because peptides are involved in a wide range of important physiological processes ! Triggers ovulation Triggers “let-down” of milk; stimulates uterine contraction Regulates plasma Ca2+ and phosphate Promotes thyroid hormone production Regulates digestion Increases water reabsorption mediates transient vasodilation Therefore Amino acids can make bonds and generate peptides and proteins That can make interactions with receptors, others proteins, fuctions controller, structures… Avanços em Engenharia de Proteínas e Peptídeos Aplicações Avanços em Engenharia de Proteínas e Peptídeos Fluorophors Fluorophor Excitation Emission Wavelength* Wavelength* Abz 320 nm 420 nm (2-Aminobenzoyl or Anthraniloyl) N-Me-Abz 340 360 nm 440 450 nm IN Methyl-anthronitoyl) AFC 395 nm 495 nm (7-Amido-4- 400 nm 505 nm trilvoromethylcournarin) AMC 3€0 nm 440 nm (7-Amido-4-melhylcoumarin) 380 nm 460 nm 380 nm 140 nm 380 nm 460 nm EDANS 340 nm 490 nm (5-(2-Aminoethyl)- aminoJnaphihalene-1-sulfonic acid) FITC 490 nm 520 nm (Fluorescein isolhioeyanate) AMBNA 335 nm 410 nm (4-Mathoxy-B-naphthylamide) 340 nm 425 nm 340 nm 430 nm 350 nm 440 nm Mca 325 nm 392 nm ((7-Methoxycoumarin-4-yacety)) BNA 320 nm 420 nm (6-Naphthylamide) 340 nm 410 nm Tp 280 nm 360 nm (Trvptophan) Avanços em Engenharia de Proteínas e Peptídeos Fret - Fluorescence Resonance Energy Transfer Acceptor absorption Donor JA) fluorescence Wavelength (A) Donor Abz (2-Aminobenzoy] or Anthraniloyl) Abz (2-Aminobenzoyl or Anthraniloyl) Abz (2-Aminobenzoyl or Anthraniloyl) Abz (2-Aminobenzoy! or Anthraniloyl) N-Me-Abz (N-Methyl-anthraniloy) EDANS (5-12-Aminocthyamino] naphthalene-1 -sulfonic acid) FITC (Fluorescein isothiceyanate) Mca ((7-Methoxycoumarin-4 -yljacetyl) Trp (Tryptophan) Acceptor (Quencher) Dnp (2.4-Dinitropheny)) EDDnp (N-(2,4-Dinitrophenyethylenediamine) p-Nitro-phenylalanine 3-Nitro-tyrosine Dnp (2.4-Dinirophenyl) DABCYL (4(4-Dimethylaminophenylazo)benzoyl) Dnp (2.4-Dinirophenyl) Dnp (2,.4-Dinitrophenyl) Dnp (2.4-Dinitrophenyl) NH2 CO X X X X X Q NH CH2 CH2 NH O2N NO2 320 nm 420 nm Baixa Fluorescência Transferência de energia hidrólise NH2 CO X X 320 nm 420 nm CH2 CH2 NH O2N NO2X X X Q NH Peptide Libraries • Positional Scanning Library • Solid Phase Library – One bead one compound • PNA – Peptide Nucleic Acid Avanços em Engenharia de Proteínas e Peptídeos PNA- Peptide Nucleic Acid HN H/N-Ace-Linker e “o e é e Ac-N ES OD ecran R o o o e e e Aa LSUS O DrRo e cce in º o e e e Ac-le-Glu-Pro-Asp-Acc-Linker e e e o e e Challenging & costly synthesisLess accumulation in tissues Cleared from body quicklyBiological & chemical diversity Risk of immunogenic effectsOften very potent Solubility challengingLower toxicity Difficult delivery; challenge to transport across membranes Minimization of accumulation in tissues Less stableLittle unspecific binding to molecular structures other than desired target Injection requiredHigh specificity Low oral bioavailabilityHigh activity DisadvantagesAdvantages Making Pre Formulations synth sis Peptidemimetics How reduce these Disadvantages ? What is Peptidemimetic ? Molecule able to show the same biological activities and structure-activity relationships as the native peptide Imagine a conversation between a small molecule and a peptide on a make-believe pharmacological playground. The small molecule would tout its virtues of small size, low price, oral availability, ability to cross membranes, and straightforward synthesis. The peptide would respond: "True, I may be bigger, more expensive to synthesize, and less stable than you. I may clear faster from the body and usually need to be injected rather than swallowed as a pill. But I can be much more potent, show higher specificity, and have few toxicology problems. I also don't accumulate in organs or face drug-drug interaction challenges like you do. So there." C&EN, 2005;Volume 83, Number 11 pp. 17-24 Avanços em Engenharia de Proteínas e Peptídeos Captopril Bothrops jararaca NpGlu-Trp-Pro-Arg-Pro-Gln-Ile O N COOH O N COOH NH2 O N COOH HOOC O N COOH SH teprotide Ala-Pro captopril The process leading to the discovery of captopril Nature Reviews Drug Discovery 4, 185-186 (2005 SH N O COOH N H N O COOH O O - N H N O COOH O O N N O COOH NH2 O O - Zn coordination Captopril ACE inhibitor Proline Carboxylate Required Remove thiol Add phenyl Make ester Remove thiol Use Lys Instead of Phe In Vivo Hydrolysis Enalaprill ACE inhibitor Lisinopril Evolution of ACE Inhibitors Structure Activity Relationship (SAR) • There was suggestions that His6 residue was required for receptor recognition • Agonist activity required the phenyl ring of the Phe8,the hydroxyl group of Tyr4, and the C-terminal carboxylate H2N-Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-CO2H The presence of biphenyl group enhanced the oral activity, but the presence of a linker chain between the two phenyl moieties reduces the activity. The acidic isostere imparts highest activity to the drug molecule when present at the ortho position. A short alkyl chain at the 2 position of imidazole or the fused imidazole ring is needed for efficient binding to the receptor. The imidazole ring is required as an acceptor in a hydrogen-bonding interaction with the receptor. Avanços em Engenharia de Proteínas e Peptídeos Others examples Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile- Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu- Ile-His-Thr-Ala-Glu-Ile-Arg-Ala-Thr-Ser-Glu-Val-Ser-Pro-Asn-Ser- Lys-Pro-Ser-Pro-Asn-Thr-Lys-Asn-His-Pro-Val-Arg-Phe-Gly-Ser- Asp-Asp-Glu Ostabolin C (hPTH-(1-31)NH2) A droga encontra-se em fase clínica 2 e será testada em 250 pacientes. Calcif Tissue Int. 1996 Feb;58(2):81-7 Amylin Lagarto Gila monster Deserto do Arizona Heloderma suspectum John Eng (Veterans Affairs Medical Center) Ziconotide 2004 o FDA aprovou PRIALT (ziconotide intrathecal infusion) para pacientes com dor crônica severa mau so so Avanços em Engenharia de Proteínas e Peptídeos 1881 Fracionamento do veneno 200 300 400 500 min ER Daio lido Au DD DI Sr SR GB e Go EB RTº (min) Molecular mass(es)º (Da) ua 1137.38, 1534.52, 203.52, 2102.88º 263 590.24, 658.33, 791.34, 1786.86, 191262 286 1233.66 288 730.35", 866.40", 973.50, 1913.62, 1922.82" 30.6 1192.54, 1829.79, 198504 34 905.38, 987 48", 1402.74º 23 107256 3737 1001.52º 555 3360.90, 3921.13, 4297.35, 5013.47 500 3446.72 64 1219263 =0.19, 12238.62 = 0.25, 12311.27 = 0.42, 12368.23=0.98 ses 1179463 0.33 58 11794.86 = 0.67 725 9088.33 = 0.94, 12533.19 + 1.44, 1343456 + 103 The bold data relates to the molecule being described in the paper. * LC-MS retention time according to Section 2.5 of a peak (or peaks) which intensity was, at least, 10% of the full scale. * standard error for molecular mass was calculated when there were, at least two, different charge states of the same molecule, otherwise measured molecular mass is presented * Orpotrin. ” Molecular masses that may correspond to other intemal frag- ments of CK, within the 0.03 Da mass. Avanços em Engenharia de Proteínas e Peptídeos Análise por MALDI-TOF 1n0z046 1,100 1,050 1,000 é E a 3 Intensity BBBEBRBESESRS ani As ich Hm it d 4 a ) 910 820 930 940 950 980 S70 SBD 990 1,000 1,010 1,020 1,030 1,040 1,050 1,080 1,070 1,080 1,080 1,100 miz Fig. 2 - MALDI-TOF spectrum of purified Orpotrin. Sequenciamento por Q-Tof E [=| E S E [= ED [- o n Ee] d= o eins 3 [-m o = [e] = o R e En =) E uu — Cn a [=] O E E es 1: TOF MEME 501.60E5+ 250605AD MaxEnt3 65 [Ev6590,1150,En1] (0.050,200.00,0:200,1400.00,2Cmph a 108562 Pai fz E Ê Be gs =maeceecancencesoncanocecanoeccemomego nã = É 1000 1050 350 400 so 950 550 600 650 70 750 db 850 500 450 150 200 250 300 100 50 Fig. 3 - Representative CIF spectrum of purified Orpotrin performed in a Q-TOF Ultima API (Micromass). b and y series are presented above the profile and the sequence is annotated using amino acids one-letter code. Avanços em Engenharia de Proteínas e Peptídeos Avanços em Engenharia de Proteínas e Peptídeos mau (B) | 1215.62 ne 100 140 120 so 100 so 80 60 “0 Phypo Xa 40 T 20 20 o 0 10.0 200 30.0 40.0 min Fig. 1 - RP-HPLG profile of P. hypochondrialis pooled crude skin secretions after solid phase extraction, performed according to Section 2. The arrow indicates fraction containing Phypo Xa, comprised between the thick lines. Insets: (A) MALDI-TOF/ MS profile of the purified peptide and (B) zoomed MALDI-TOF/MS of the peptide presenting the isotopic distribution. Measured m/z values are typed besides the ions. Avanços em Engenharia de Proteínas e Peptídeos A RA a Dia Ta AD bi Et Peptide Name MW (Da) Source sequence <EFRPSYQIPP Fhypo Xa 1214.6 Phyllomedusa hypochandrialis <ESWPGPNIPP BPP-10a 1074.5 Bothrops jararaca <ENWPRPQIPP BFP-10b 1214.6 Bothrops jararaca <ENWPHPQIPP BPP-10c 1185.6 Bothrops jararaca <ERWPHLEIPP - 1254.6 Crotalus d. terrificus «<EGLPPRPIPP - 1053.6 Agkistrodon halys blomihoffi <EwP..qlPP - - Consensus” <E represents pyroglutamic acid. a Calculated by Clustal W [46] Table 2 = Vascular effects of the IV administration of a TUR E Drug Time” Omin Smin (MAP 30min (MAP (Control") decrease!) decrease) 0.9% Nacl 100 + 20 5+6 ND: Phypo Xa 50 pg 100 + 20 3245 25+3 Phypo Xa 100ng 100420 73+6 51+10 Captopril 43 ug 100 + 20 5949 N.D. * Time after 500 ng BK administration. » Normalized data. * Mean arterial pressure. * Percentual decrease, related to the control group; n =4. * Not determined. Avanços em Engenharia de Proteínas e Peptídeos obal conserto (cyolzation). straints (specialized amino Xcray Ive a and o 3D a DR] design Peptidomimetics Non-peptidic Peptide Analogs Peptides Natural peptide Deletions from the natural sequences Substitution of amino acids in the natural sequences Stereochemical substituitions with D-amino acids Introduction of side-chain conformationally restricted amino acids or proline analogs Isosteric and other substituitions with non proteinacious amino acids Peptide bonds surrogates: N-alkylated peptides, Cα tetrasubstituited amino acids, dipeptides, isosteric peptide bond replacement Topological modifications of the peptide main chain: Introduction of β-amino acids or γ-amino acids, retro-inverso peptides, ureine or urethane peptides, insertion of aromatic spacers Combinations of side-chain and main-chain modifications Therefore with use of peptidemimetic we searching the better interaction between target and the drug. Name Avanços em Engenharia de Proteínas e Peptídeos Synthetic/modified venom peptides Voltage-Gated Ca? Severe chronic inflammatory and Granted FDA PrialtH RESNES E É Elan Corporation E co-Conotoxin MVIIA | Conus magus Channels neuropatic pain associated with | Approval (Dec. (SNX-111, Ziconotide) E) Cancarand AIDS 2004) (wyww.elan.com) Voltage-Gated Ca? ss : E p Severe chronic pain associated Amrad Corporation AM336 c-Conotoxin CVID Conus catus Chaníeis iticancer Phase ll oie tad coma) ii Chronic neuropathic pain, and ' : E SS Neuronal Nicotinic é Eai e Metabolic Pharmaceuticals ACvI a:ConotoxinVcl.1 | Conusvictoriae celolaoline cepioro Ro ofrecovery ofinjured | Preclinical (rir metabollocom au) Xen174 eConotoxin yMrA | Conus marmoreus Rorepireh Nociceptive and neuropathic pain | Phase Perunção te transporter (NET) E paca (wmyn.xenome.com) P j 1 na ri Xenome Ltd p-Conotoxin pTIA Conus tulipa a,adrenoreceptor Nociceptive and neuropathic pain | Preclinical (www. xenome.com) E rs Neurotensin Receptor Short-term management ofpost- | Completed early Cognetix Inc. GNico orlando Conta dao pinta agonist operative pain Phase | (iwww.cognetix.com) 5 q NMDA receptors Nociceptive pain and control of Cognetix Inc. Ee Nioo] Conantokin-G Conus tulipa NR2B subtype seizures in intractable epilepsy Phase l (www.cognetix.com) a e : : : TransMolecular Inc. E am TM-601 |-Chlorotoxin Leiurus quinquestriatus Cl'channel Brain tumors Phase ll nrvr.transmolecular:com) Chronic monotherapy and . . ; mis alinanesti pharmaceutical sensitizer co- a TransMolecular Inc. TM-701 |-Chlorotoxin Leiurus quinquestriatus Cl'channel administered drug cocktails for Preclinical (esutenenleasegm) cancer E Southern copperhead viper Te Thrombolytic agent Nuvelo Inc. Alimeniase brólaso (Agkistrodon contortrix) Rs and catheter occlusion Rigs (www. nuvelo.com) Pivotal Biosciences/ E Southern copperhead viper E E University of Southern donos ato (Agkistrodon contortrix) rteerin ERsiare Frei California (www.pivotalbiosci ences.com) Exenatide Exendin-4 Gila monster (Heloderma Glucagon-like peptide-1 Type-2 diabetes and related Amylin Pharmaceuticals. suspectum) metabolic disorders (www amylincom) Avanços em Engenharia de Proteínas e Peptídeos Peptomimetics or small molecular weight derivatives ' Brazilian arrowhead viper Angiotensin Converting : Granted FDA Bristol-Myers Squibb e Capote (Captonril) (Bothrops jaracusa) Enzyme (ACE) Antihpertensive! approval (www brms.com) Schering-Plough Millennium Acute coronary syndrome : Southeastern pygmy . Pharmaceuticals ma ieiinron f Platelet glycoprotein llbj | (ACS)and for patients without | Granted FDA E Integrilinº (Eptifibatide) popa lia receptor Inhibitors | ACS undergoing percutaneous approval pr A u u coronary intervention (rw scherinEplouish. com) (www millennium.com) Approved for use . Platelet giycoprotein llbj . with heparinand | Merck Aggrastatº (Tirofiban) African saw-scaled viper lllareceptor Inhibitors Acute coronary syndrome (ACS) aspirin for the: STO) treatmentofALS Neurobinlagical Industries inrine Tá ) Malaysian pit viper Heparin-induced pune nro) (Agkistrodon rhodostoma) thrombocytopenia Phase ll Uitã» a (www ntii.com) Exantar pr Tarombininhibitors | Atrialfibriliation and blood pestne a EE (Ximelagatran) clotting after orthopedicsurgey | PRIVES (wmmmastrazeneca.com) sold in Europe Delucemine(NPS 1506) Spider venom toxin NMDA biocker Protection ofbrain cells from ischaemia Phase | NPS Pharmaceuticals (www npsp.com ) Avanços em Engenharia de Proteínas e Peptídeos DEVELOPMENT No lack of hopefuls in the global peptide drug pipeline In On the market registration —, 5% Advanced 1% ú preclinical phases 56% Clinical phases 38% 2004 peptide drugs/candidates = 720 SOURCE: Frost & Sullivan C&EN, 2005, Volume 83, Number 11 pp. 17-24 INSTITUTO BUTANTAN CAT 4 Laboratório Especial de Toxinologia Aplicada www.catcepid.com.br robson@butantan.gov.br Dr Robson L. Melo Laboratório Especial de Toxinologia Aplicada