Hobaneng ha Cbl-b e le Nyeoe ea Teko e Batlang
Cbl-b (Casitas B-lineage lymphoma-b) ke E3 ubiquitin ligase e phahamisang moeli oa ts'ebetso ea lisele tsa T le lisele tsa NK., e sebetsa e le setsi sa tlhahlobo ea 'mele ea ho itšireletsa mafung eo lihlahala li e sebelisang ho thibela ho itšireletsa mafung. Joalo ka Ho shebana le Cbl-b ho immunotherapy ea mofetše (2023) oa hlalosa, ts'ebetso e phahameng ea Cbl-b e boloka lisele tse sebetsang hantle hore li se ke tsa bulela ka botlalo, e etsang hore ligase e be sehlahisoa se khahlehang empa se le bonolo ho baeloji.
Phephetso ea k'hemistri ke hore litšebelisano tse amehang tsa Cbl-b ke likhokahano tsa protein-protein le protein-phosphotyrosine., eseng nyane, lipokothong tsa lithethefatsi tsa mofuta oa peptide e melang e tlama habonolo. Hona ke boemo boo ho bona li-peptide tse koetsoeng tsa cyclic li bolokang tsona: ho koala peptide ho macrocycle ho e hlophisa esale pele hore e be sebopeho se tlamang. Ke ka lebaka leo phospho-pentapeptide Cblin (DGpYMP) le phage e nkiloeng CBLock, nanomolar peptide inhibitor sebakeng sa CBL TKB, taba. Li bonts'a hore sepheo se nkoang se sa rarolloe ke li-peptide tse bonolo li ka sebelisoa hang ha ho se ho na le meaho e thata le tsela e tiileng ea netefatso..
Ts'ebetso ea Discovery-to-Synthesis ka Mehato e Tšeletseng
Pipe e hlahisang mokhethoa oa cyclic peptide e thehiloe hantle, 'me e beha sethala bakeng sa ntho e' ngoe le e 'ngoe eo lab ea synthesis e tlamehang ho paka. Laeborari ea phage ea mefuta e fihlang ho 10¹–10¹² e hahiloe hore li-peptide tse bonts'itsoeng li ka tsamaisoa ka baesekele., e ntan'o ba khahlanong le immobilized Cbl-b ka makhetlo a 'maloa ho matlafatsa li-binders. Lintho tse kentsoeng tse ntlafalitsoeng li hlophisitsoe ho khutlisa maikutlo a tumellano, Li-hits tse holimo lia khethoa, 'me tatellano eo e kopantsoe hape joalo ka li-peptide tsa cyclic tse fapaneng - hangata ka synthesis ea peptide e tiileng. (SPSS) - pele ho tlamahano ea orthogonal le tlhahlobo ea ts'ebetso.
Joalo ka tlaleho ea cyclic peptide toolkit (2024) bontša, mohato oa makhaola-khang ha se khetho empa ke resynthesis le tlhoekiso: tatellano e ntlafalitsoeng e leshome le metso e 'meli e ile ea kenngoa ho SPPS, ebe acidified, e hloekisitsoeng ke HPLC, le ho tsebahala pele tlhahlobo efe kapa efe e ka tšeptjoa. Tlhaloso eo e le 'ngoe e bontša hore na ke hobane'ng ha litšiea tse hlano tsa tiiso e le tsa bohlokoa hakana.
Leano la ho Tsamaisa Baesekele: Geometry ke Moputso
Ts'ebetso ea bioloji ea peptide e thibetsoeng e ipapisitse le sebopeho sa selikalikoe le k'hemistri ea khokahano, eseng feela amino acid linear tatellano. Macrolactam ea hlooho-to-mohatla, selikalikoe se nang le disulfide, thioether-linked macrocycle, kapa scaffold ea CuAAC-cyclized e 'ngoe le e' ngoe e beha sebopeho se fapaneng sa mahlakore a mararo, 'me ka hona ho lekana ka tsela e fapaneng ho ea holimo ho sepheo.
Thibelo e sebelisitsoeng potolohong ea maiketsetso e tlameha ho bonts'a eo phage e bonts'itsoeng. Tsela ea tlhaho ea tlhaho e tiisa taba eo: haufi-ufi-khannoa, cyclization e khethehileng sebakeng (2024) le lilaebrari tsa cyclic peptide tse kentsoeng ka liphatsa tsa lefutso (2019) ka bobeli li hatisa hore selikalikoe se entsoeng holim'a phage ke conformation eo u batlang ho e hlahisa ho eona. Haeba tsela ea synthetic cyclization e fapana, motsoako o tsosolositsoeng e ka 'na ea se ke ea e-ba molek'hule e khethiloeng. Mona ke moo molekane ea nang le boiphihlelo ba cyclic peptide synthesis - setsebi se kang MOL Changes, eo tloaelo ea cyclic peptide synthesis e ka tsamaisana le thibelo e khethiloeng, laola tatellano e thata ea hydrophobic le ho bokellana, ebe u khetha tsela e nepahetseng ea ho koala lesale, ke motheo oa ho otla ka botšepehi.
Netefatso ya Tatelano: Ho netefatsa seo o se entseng
Li-peptide tsa cyclic ke tse ling tsa limolek'hule tse thata ka ho fetisisa ho tatellano. Ha ho na mahala N- kapa C-terminus, ho senyeha ha khale ha Edman ha ho khonehe, NMR e hloka thepa e ngata ho feta eo skrine e atisang ho e hlahisa, 'me tandem mass spectrometry e arola selikalikoe lintlheng tse ngata, qaka tlhaloso. Joalo ka thuto ea multistage mass spectrometry (2011) oa hlalosa, ho netefatsa tatelano e reriloeng le topology ka hona ho hloka mekhoa ea ho arola li-orthogonal - mekhoa ea ring-cleavage, MS/MS ea mekhahlelo e mengata, kapa tlatsetso digests - ho abela tlhamo le kgokahanyo ntle ho pelaelo.
Netefatso ea tatelano ke phapang lipakeng tsa peptide e lumellanang le data ea ntlafatso le e tšoanang le eona feela.. E kopantsoeng hampe, fokotsehile, kapa mefuta e epimerized e ka tsamaisa ts'ebetso eo tlhahlobo ea tlase ea noka e amahanyang le sepheo se reriloeng. Netefatso e kenyelletsang boitsebiso ka bobeli (bongata bo boholo) 'me netefatso ea tatelano ke keiti ea tokollo e ke keng ea buisanoa pele ho tlhaloso efe kapa efe ea baeloji.
Likhetho tsa ho ngola: Tag e Fetoha, Eseng Sesebediswa
Peptide Synthesis Lits'ebetso tse ngata tsa ho netefatsa li hloka molamu o bonoang, 'me likhetho tse peli tse tloaelehileng ke biotin le fluorophore tse kang FITC. Biotin e etsa hore motho a se ke a tsamaea holim'a streptavidin bakeng sa ho hula fatše le liteko tsa tlhōlisano., ha FITC e ts'ehetsa polarization ea fluorescence kapa lithuto tsa ho nka. Empa label ha e nke lehlakore. Letlapa le leholo le ka fetola tlamahano, ho qhibiliha, permeability, esita le kamano e bonahalang eka ke ea macrocycle e nyenyane, kahoo ho tlameha ho bonts'oa analoge e ngotsoeng ho boloka boitšoaro ba peptide ea motsoali pele tlaleho ea eona e ka tšeptjoa.
Ena ke mohloli oa khale oa kholiseho ea bohata ho netefatso ea hit: kamano e khahlang e lekantsoeng moahong o tšoailoeng e ka bonts'a litlamorao ho fapana le boitlamo ba 'nete. Ho khetha lik'hemik'hale tse nepahetseng - ho kenyelletsa bolelele ba sehokelo le sebaka sa sehokelo - le ho netefatsa lengolo khahlano le taolo e sa ngolisoang ho bohlokoa.. Phatlalatso ea phetoho ea molekane oa synthesis, pharalla biotinylation ea peptide le fluorescent (FITC, Cy) ho ngola, e fetola sena ho tloha papaling ea papali ea chelete ho ea tekong e laoloang.
Tlhahlobo ea Bohloeki: Ho Arola Hit Ho Baahelani ba Lona
cyclic peptide synthesis ha e hlahise mofuta o le mong. Cycization e sa fellang e siea li-precursors tse tšoanang, oligomerization e hlahisa dimers, le isomerization kapa epimerization e hlahisa lihlahisoa tse amanang empa tse ikhethang - tseo kaofela li ka khopamisang ho bala ha tlhahlobo.. Lintho tse hloekileng tse phahameng, e netefalitsoe ke HPLC mme e tšehetsoa ke profil e felletseng ea litšila, e fokotsa kotsi ea hore ts'ebetso e hlokometsoeng e tsoa ho litšila ho fapana le macrocycle e reriloeng.
Bakeng sa motho ea etsang liqeto o fumana li-peptide tsa maemo a holimo, sena se fetolela tlhokahalo ea konkreite: batla chromatogram, eseng feela peresente ea bohloeki. Bohloeki ba HPLC ka holimo ho 95–98%, ka data e tsamaeang le MS le litokomane tse ikhethileng haholo, ke sona se etsang hore tatellano ea tšebetso ea sebopeho e be le moelelo le ho ikatisa ka bongata. Ho hloeka ha se tloaelo; ke heke e thibelang ho otloa ha 'nete hore e se ke ea patoa - kapa ho felloa ke moea ke - baahisani ba eona.
Boitsebiso bo Lokisitsoeng ke Tekolo: Bopaki ke Ho Ikatisetsa
Matlafatso ea Phage e paka hore mokhethoa a ka tlama ha a bonts'a karoloana ea vaerase; ha e pake hore molek'hule e tsosolositsoeng e sebetsa sebakeng se hloekileng, tekolo e emeng. Lintho tse itokiselitseng tlhahlobo li bolela hore motsoako o fihla ka bongata bo lekaneng, ka mokhoa o nepahetseng oa letsoai, e se nang li-counterions tse sitisang le endotoxin, le ka litokomane tse lekaneng hore lab e ncha e ka hlahisa sephetho ntle le ho ntlafatsa k'hemistri.
Mona ke moo sephutheloana sa tlhahlobo se fetohang tekanyetso ea tokollo ho e-na le ho nahana ka morao. Endotoxin, boima ba mmele, pH, mongobo, litaba tsa counterion, 'me sterility ke litšobotsi tse khethollang hore na peptide e itšoara ka tsela e tšoanang ka Labone joalokaha e entse ka Mantaha - le hore na sephetho se tla tšoarella lenaneong le shebaneng le taolo.. E hlahisitsoe tlas'a a Sehlopha 100 tikoloho e laoloang ke boleng e nang le traceability e felletseng, lisebelisoa tse lokiselitsoeng ho etsa liteko li fetola tatellano e ts'episang hore e be ntho e ka itšireletsang, lintlha tse ka phetwang tsa data.
Lethathamo la netefatso bakeng sa Lihits tsa Peptide tse Khatelitsoeng
- Netefatsa tsela ea synthetic cyclization e lumellana le tšitiso e khethiloeng holim'a phage.
- Netefatsa boitsebiso le tatelano ka orthogonal mass spectrometry, eseng boleng bo le bong ba boima.
- Netefatsa label efe kapa efe ea biotin kapa fluorophore khahlano le Li-Peptide tsa Synthetic taolo e sa ngolwang pele o tshepa se baliloeng.
- Batla bohloeki ba HPLC le boemo bo felletseng ba litšila, hammoho le lintlha tse tsamaeang le MS. Tlhahiso ea Peptide
- E hloka data e lokiselitsoeng ho lokolloa: counterion, endotoxin, boima ba mmele, pH, le nyopa.
- Netefatsa ho ikatisa ho hongata ho isa ho lotho pele o phahamisa ho tloha tlhahlobong ho ea ho ntlafatso ea lead.
Key Takeaway: Phage-display hit e fetoha moetapele feela ka mor'a ho kopanya le ho bonts'a hore peptide e nchafalitsoeng ea cyclic e tsamaisoa ka nepo., tatellano-e tiisitsoe, e ngotsoe hantle, bohloeki bo phahameng, ebile e ka ikatisa. E 'ngoe le e 'ngoe ea litšiea tse hlano ke heke e ikhethileng e thibelang lintho tsa khale hore li se ke tsa kena pompong ea ho sibolloa ha lithethefatsi..
Meeli ea Lense ena ea netefatso
Litlhahiso tse peli li boloka moralo ona o tšepahala. Ea pele, ha ho na pampiri e bonts'itsoeng ke lithaka ea Cbl-b cyclic peptide phage hajoale e kopanyang tatellano e felletseng, data e tlamang, le mochine sebakeng se le seng; Phallo ea mosebetsi e kaholimo ke mokhoa oa mokhoa oo boithuto bo joalo bo ka o latelang le hore peptide e haufi ea Cbl-b e sebetsa. (Cblin, CBLock) e se e le mehlala. Ea bobeli, netefatso ea katleho e ikhethang litekong tse tlamang ha e so ka e bolela ka thuso ea kalafo - Thibelo ea Cbl-b in vivo e na le kotsi ea 'nete ea autoimmunity., kahoo ts'ebetso ea bohlokoa e tlameha ho bontšoa ka cellular le, qetellong, mehlala ea liphoofolo pele ho loto e nkoa e le lithethefatsi.
Mehato e Latelang
Phapang pakeng tsa skrine e ntle le moemeli ea molemo oa lithethefatsi e etsoa qeto ho tloha phage, k'hemistri le sebopehong se fetolang tatellano hore e be e netefalitsoeng, molek'hule e itokiselitseng liteko. Haeba u tsamaea, li-peptide tsa cyclic li otla ka synthesis le netefatso, molekane ea nang le boiphihlelo ba peptide a ka koala sekheo seo - bua le sehlopha sa tekheniki ka tatellano ea hau 'me u fumane tlhahlobo ea bokhoni pele u etsa tekanyetso ea hau ea tlhahlobo..
