Kev Tshawb Fawb Ntawm Novel STING Inhibitors Raws Li Cov Qauv Ntawm Tus Nas STING Agonist DMXAA
Nov 27, 2023
Abstract:
Lub stimulator-of-interferon-gene (STING) protein yog koom nrog hauv kev tiv thaiv kab mob. Cov tshuaj DMXAA (5,6-dimethylxanthenone-4-acetic acid) tau ua pov thawj tias muaj zog murine-STING (mSTING) agonist tab sis muaj kev cuam tshuam me ntsis rau tib neeg-STING (hSTING). Nyob rau hauv daim ntawv no, peb kos raws li kev sib piv ntawm cov qauv siv lead ua sib txawv thiab cov kev sib raug zoo ntawm cov protein-ligand kev sib raug zoo los soj ntsuam qhov kev xav tias cov tshuaj tsim ntawm DMXAA variants muaj peev xwm hloov STING agonists mus rau inhibitors. Raws li peb qhov kev tshawb pom yav dhau los ntawm ob DMXAA analogs, 3 thiab 4 (ob leeg tuaj yeem khi rau STING), peb tau ua kom zoo dua lawv thiab ua kom cov khoom siv tshiab, raws li. Hauv kev txheeb xyuas kev sib txuas, peb pom cov khoom sib txuas 11 thiab 27 los sawv cev rau STING binders uas zoo tshaj rau cov qauv qub thiab sib tham txog cov qauv-kev sib raug zoo. Tag nrho cov hom phiaj sib txuas tsis ua haujlwm hauv kev soj ntsuam ntawm tes rau kev tshuaj xyuas ntawm STING agonistic kev ua haujlwm. Ua tsaug, peb tau txheeb xyuas 11 thiab 27 raws li STING inhibitors nrog micromolar kev ua haujlwm hauv ob qho tib si hSTING thiab mSTING txoj hauv kev. Tsis tas li ntawd, 11 thiab 27 inhibited qhov induction ntawm interferon thiab inflammatory cytokines qhib los ntawm 20 3 0 -cGAMP yam tsis muaj cytotoxicity. Cov kev tshawb pom no rhuav tshem cov kev xav tsis zoo uas DMXAA muab cov qauv tsim tshwj xeeb rau STING agonists thiab qhib ntau txoj hauv kev los tsim cov STING agonists tshiab lossis inhibitors.
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Ntsiab lus:
STING; DMXAA; hom kev xaiv; STING agonist; STING inhibitor
1. Taw qhia
Lub stimulator-of-interferon-gene (STING) yog ib qho tseem ceeb taw qhia molecule rau intrinsic tiv thaiv kab mob, feem ntau kho cytoplasmic DNA-induced natural tiv thaiv teb [1]. Thaum lub DNA receptor cyclic guanosine-adenosine phosphate synthase (cGAS) kuaj pom intracellular ob-stranded DNA, cGAS catalyzes cov synthesis ntawm 2 0, 30 -cGAMP (Daim duab 1), cyclic dinucleotide (CDN) muaj peev xwm. ntawm kev khi ncaj qha rau thiab ua kom STING ntawm endoplasmic reticulum [2–4]. Thaum ua kom muaj zog, STING cov ntaub ntawv sib sau ua ke uas tau txais cov dej ntws mus rau TANK-binding kinase 1 (TBK1) thiab khi interferon regulatory factor 3 (IRF3), uas phosphorylates IRF3, activates IRF3 dimers rau hauv lub nucleus, induces hom I interferon (IFN) qhia thiab pib. kev tiv thaiv kab mob interferon [1]. Ntau cov kev tshawb fawb tau pom tias STING koom nrog hauv cov kab mob ntawm ntau yam kab mob thiab qhov stimulation ntawm STING induces lub cev tiv thaiv kab mob rau cov kab mob thiab cov qog nqaij hlav; Txawm li cas los xij, tsis ua raws li cov teeb meem mob ntev ua rau muaj kab mob autoimmune thiab inflammatory [5–8]. Vim yog lub luag haujlwm tseem ceeb ntawm STING hauv kev tswj hwm kev tiv thaiv hauv lub cev, ntau pab pawg tau pib tsim STING agonists lossis inhibitors.
Daim duab 1. Tus neeg sawv cev STING modulators
Kev tshawb fawb ntawm thawj tiam STING agonists tau tsom mus rau kev hloov kho cov qauv ntawm CDN analogs kom hla qee qhov kev txwv ntawm CDNs endogenous, xws li cov membrane permeability tsis zoo thiab raug rau hydrolysis los ntawm phosphatase [9,10]. Cov molecule STING agonist 5,6-dimethylxanthenone-4-acetic acid (DMXAA), uas tau qhia cov lus cog tseg kho mob rau cov qog nqaij hlav hauv cov qauv nas, tuaj yeem tiv thaiv qhov tsis zoo ntawm CDN derivatives [11]. Txawm li cas los xij, cov tshuaj ua tsis tiav hauv tib neeg kev sim tshuaj [12]. Nws tau lees paub ntxiv tias DMXAA xaiv khi rau murine STING (mSTING) es tsis yog rau tib neeg STING (hSTING) [13,14], uas cuam tshuam cov peev txheej kho mob ntawm DMXAA hauv tib neeg. 10-Carboxymethyl{11}}acridinone (CMA) yog lwm tus neeg sawv cev murine-specific STING agonist siv los ua hom muaj zog I IFN inducer rau kev kho tshuaj tua kab mob thaum ntxov 1970s [15]. Yog li ntawd, hom kev xaiv yog qhov tseem ceeb hauv kev txhim kho cov me-molecule STING agonists. Ntau yam tshiab-skeleton hSTING agonists tau coj los rau hauv kev pom los ntawm kev siv zog ntawm cov kws tshawb fawb, suav nrog diABZI, SR717, MSA02, thiab lwm yam [16–19].
cistanche cov txiaj ntsig rau txiv neej-ua kom muaj zog tiv thaiv kab mob
Ntau cov pov thawj qhia tau hais tias STING hyperactivation yog txuam nrog autoinflammatory thiab autoimmune kab mob. Nws yog ib qho tsim nyog los txwv qhov kev ua kom ntau dhau ntawm STING signaling txoj hauv kev, uas qhia txog qhov muaj peev xwm ntawm STING inhibitors. Nyob rau hauv sib piv rau STING agonists, txoj kev loj hlob ntawm STING inhibitors tseem nyob rau hauv nws cov me nyuam mos, txawm li cas los xij, thiab tsis muaj cov neeg sib tw tshuaj tau nkag mus rau kev tshawb fawb soj ntsuam. Cov paub STING inhibitors suav nrog ob hom kev sib txuas: kev sib tw antagonists (SN-011 thiab Merck-18) thiab covalent inhibitors (H151) [20–22]. Covalent STING inhibitors yav dhau los cuam tshuam nrog Cys88/91 lossis Cys91 uas nyob hauv N-terminal transmembrane domain ntawm STING sab nraum CDN-binding hnab tshos [22]. Tsis ntev los no, ob pab pawg tau ua tiav tau tshaj tawm ob hom qauv ntawm STING antagonists uas nyob hauv 20 3 0 -cGAMP khi hnab ris los tiv thaiv kev ua kom ntawm 20 3 0 -cGAMP, qhia txog qhov cuam tshuam ob sab hauv CDN-binding hnab tshos [20 , 21] ib.
Ntawm no, peb muab qhov kev xav tshiab. Peb xav tias hauv qab hnab tshos qhov twg DMXAA thiab CMA nyob rau hauv pab txhawb kev txhim kho ntawm STING inhibitors raws li peb cov ntaub ntawv sib sib zog nqus mining thiab kev sib piv ntawm cov ntaub ntawv co-crystal qauv ntawm STING cov proteins. Peb txoj kev tshawb fawb yav dhau los tau tshaj tawm txog CMA thiab DMXAA analogs thiab txheeb xyuas cov khoom sib txuas 3 thiab 4 uas khi rau hSTING tab sis muaj zog cellular tsis muaj zog [23] (Daim duab 2).
Daim duab 2. Cov qauv tshuaj lom neeg ntawm cov khoom sib txuas 3 thiab 4 nrog cov ntaub ntawv ntawm lawv cov dej num lom neeg. EC50 qhov tseem ceeb ntawm cov tebchaw 1-4 tau ntsuas los ntawm STING cov xov tooj ntawm tes, thiab kev khi IC50s tau ntsuas los ntawm STING cov khoom sib tw khi.
Nyob rau hauv daim ntawv no, hais txog tus tsim ntawm STING inhibitors kom nrhiav tau cov muaj zog binding neeg ua hauj lwm nrog ntau cov neeg nyob rau hauv lub hnab tshos hauv qab, peb tau ua cov txheej txheem optimization rau 3 thiab 4, feem. Tom qab ntawd, peb tau ua qhov kev tshawb fawb SAR raws li kev sib tw-kev txheeb xyuas kev sib tw. Hauv kev ntsuam xyuas bioactivity, cov tebchaw 11 thiab 27 tau ua raws li cov broad-spectrum binders thiab nthuav tawm micromolar qib ntawm STING inhibitory kev ua haujlwm hauv ntau lub hlwb. Nyob rau hauv cov nqe lus ntawm docking qauv, ob compound 11 molecules tuav hSTING nyob rau hauv ib tug inactive "qhib" conformation, yog li kev sib tw inhibiting endogenous 20 3 0 -cGAMP binding, uas zoo ib yam li cov qauv siv lead ua ntawm Merck-18 thiab cov qauv docking. ntawm SN-011 [20,21].
2. Cov txiaj ntsig
2.1. Tsim Lub Tswv Yim ntawm DMXAA Derivatives li STING Inhibitors
2.1.1. Kev txheeb xyuas qhov chaw tsim qauv rau STING Inhibitors los ntawm kev sib piv cov qauv siv lead ua sib txawv
Lub apo-protein ntawm STING ligand binding domain (LBD) crystallized raws li ib tug symmetrical dimer nyob rau hauv lub non-ligand, uas yog proven tsis yog tshwm sim los ntawm lub gel filtration thiab analytical ultracentrifugation tsom xam [24–26]. Lub STING dimer txais kev sib haum xeeb zoo li npauj npaim nrog lub ligand khi qhov chaw nyob ntawm qhov sib cuam tshuam ntawm ob lub monomers. Ua ntej, daim ntawv no tshuaj xyuas cov txheej txheem tsim kho thiab ua kom muaj zog ntawm hSTING thiab mSTING cov proteins, sib piv qhov sib txawv ntawm lawv, nrog rau nthuav tawm qee qhov kev nkag siab zoo. Rau cov qauv apo, lub ntuj conformation ntawm ob qho tib si proteins qhia tau hais tias lub mSTING yog ntau concentrated tshaj lub hSTING (Daim duab 3). Shih et al. Kev ua haujlwm molecular dynamics (MD) simulations los kawm txog qhov sib txawv ntawm hSTING thiab mSTING, qhia tias hSTING nyiam qhov qhib-inactive conformation, thiab mSTING nyiam qhov kaw-active conformation txawm tias tsis muaj ligand khi [27].
Thaum khi rau 20, 30 -cGAMP, kev hloov pauv ntawm hSTING yog los ntawm qhib (apo-protein, lateral nrug ~ 57 Å) kom kaw (cGAMP-bound protein, lateral nrug ~ 40 Å), whereas qhov opposite yog muaj tseeb rau mSTING (29 Å rau 40 Å). Cov qauv ntawm apo-hSTING txawv heev ntawm apo-mSTING, tab sis cov qauv ntawm STING complexed nrog 20, 30 -cGAMP sib tshooj. DMXAA, CMA, thiab cGAMP yog STING agonists nrog ntau yam qauv scaffolds, tab sis lawv cov kev ua kom haum raws li kev khi rau mSTING cov proteins kuj tseem zoo ib yam (Daim duab 3). Cov xwm txheej tau piav qhia saum toj no ua pov thawj tias cov txheej txheem stimulation ntawm hSTING thiab mSTING yog qhov sib txawv heev; Txawm li cas los xij, cov kev hloov pauv tau qhib los ntawm STING yog, txawm li cas los xij, tsis tu ncua, ywj siab ntawm hom kev xaiv thiab hom kab mob agonist (thaum txwv rau STING agonists uas coj txog kev hloov pauv). Lub hnab ntim cov khoom sib txawv ntawm cov khoom siv lead ua sib txawv feem ntau yog convergent (~ 300 Å3, Table S1), uas yog ntxiv pov thawj ntawm kev ruaj ntseg ntawm STING protein ua kom muaj kev sib haum xeeb.
Daim duab 3. Kev sib piv ntawm apo- thiab compound-bound STING crystal lug. Lub mSTING protein yog nplej (nyob deb sim nrog kab liab), PDB code: 4KC0 (apo), 4LOJ (bound-cGAMP), 4LOL (bound-DMXAA), thiab 4JC5 (bound-CMA)
Thib ob, raws li cov kev tshawb pom saum toj no ntawm kev ua kom muaj kev ruaj ntseg, peb tau saib xyuas cov qauv siv lead ua ntawm cGAMP, DMXAA, CMA, SR717, thiab MSA02, uas yog kaw-conformational STING agonists, los tshawb txog txoj hauj lwm ntawm cov txheej txheem sib txawv ntawm cov agonists hauv lub STING protein khi qhov chaw. Lub hnab ntim cov protein tuaj yeem muab faib ua ob thaj tsam: hauv qab hnab tshos thiab lub hnab tshos sab saum toj, tom qab kev faib tawm ntawm STING agonists hauv lub hnab ntim cov protein (Daim duab 4a). Tsuas yog rau mSTING agonists DMXAA thiab CMA tag nrho ntawm lub hnab tsho hauv qab, lwm tus agonists tuaj yeem qhib txoj hauv kev hSTING thiab pw hauv cheeb tsam sab saud. Peb mam li qhia txog kev sib cuam tshuam ntawm tsib STING agonists nrog cov amino acid residues khi rau STING protein (hauv 5 Å ntawm ligand; daim duab S1), uas qhia tias cov amino acids tseem ceeb suav nrog R238, Y167, R232, S162, T263, thiab T267. (cov mSTING sib thooj cov seem uas yog tus lej rho tawm ib). Sib piv cov seem seem mus rau lub hnab ris pom tias T267, T263, thiab S162 pw hauv qab hnab ris; lub hnab tshos sab saum toj muaj R238, R232, thiab Y167 (Daim duab 4b). Raws li tau tshaj tawm, R238, Y167, thiab R232 yog qhov tseem ceeb rau agonist khi rau STING, tshwj xeeb tshaj yog R238 (cov protein mSTING sib raug rau R237), qhov kev hloov pauv uas yuav ua rau muaj kev ruaj khov (ligand binding) [28–30]. Che et al. kuj tau qhia tias qhov tseem ceeb-residue R238 dominates qhov khi ntawm DMXAA, thiab cov ntsiab lus hloov pauv (S162A / E260I) tuaj yeem txhim kho kev sib cuam tshuam ntawm R238 nrog DMXAA los ntawm MD simulations [31].
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Txhawm rau tuav lub hnab ntim cov protein mSTING, DMXAA lossis CMA tau txais ib qho kev sib khi tshwj xeeb uas ob lub me me-molecule agonists khi rau ib qho mSTING homodimer. Lub DMXAAs ruaj khov rau hauv qab hnab tshos los ntawm hydrogen bonds ntawm pawg keto thiab T266 sab saw (hSTING sib raug rau 267), thaum pawg carboxylate cuam tshuam nrog R237 thiab T262 sab chains (Daim duab 5a). Kev sib cuam tshuam ntawm DMXAA nrog R237 (tsuas yog qhov tseem ceeb amino acid nyob rau hauv lub hnab tshos sab saum toj) yog qhov tseem ceeb rau kev ua haujlwm mSTING. Ib qho kev nthuav dav tshaj plaws yog tias DMXAA tsuas tuaj yeem ua rau R237 ntawm symmetric monomeric proteins, piv txwv li, DMXAA (molecule A) cuam tshuam rau R237B (Daim duab 5a). Vim tias DMXAA zaum hauv qab, qhov kev ncua deb ntawm molecule A thiab R237B yog 3.06 Å, thaum qhov kev ncua deb rau R237A yog ntau dua 5 Å (Daim duab 5b). Tau txais txiaj ntsig los ntawm lub hnab ntim ntau dua ntawm mSTING protein, DMXAA cuam tshuam nrog R237 ntawm symmetric mSTING monomer tab sis tsis muaj txoj hauv kev rau DMXAA los koom nrog ib qho ntawm R238 hauv conformationally qhib apo-hSTING protein (Daim duab 3). Li no, lub hnab tshos txoj hauj lwm ntawm DMXAA impairs lub stimulation ntawm txoj kev hSTING. Tsis ntev los no, Merck, kev tshoov siab los ntawm 2: 1 binding ratio ntawm DMXAA, tau tshawb pom STING antagonists muaj peev xwm tuav lub hnab ntawv khi [21]. Lub crystal complex ntawm Merck 18 thiab hSTING cov protein qhia tau hais tias cov compound feem ntau nyob hauv qab lub hnab ntim cov protein thiab cuam tshuam nrog S162, T263, thiab T267, tag nrho cov uas phim cov khoom ntawm DMXAA (Daim duab 5c). Los ntawm qhov no, cov kev xav tau raug tsim tawm: lub hnab tshos sab saum toj yog cov kab noj hniav zoo heev rau kev tsim STING agonists; hauv qab hnab tshos yog lub zes tsim nyog rau inhibitors, thiab cov qauv ntawm DMXAA los yog CMA yog optimized kom muaj peev xwm los ua ib tug STING antagonist.
Daim duab 4. Kev faib tawm ntawm STING qhov chaw khi thiab faib nrog cov amino acids tseem ceeb. (a) Qhov chaw ntawm ob cheeb tsam hauv STING monomer. Lub hnab tsho hauv qab yog xim magenta thiab lub hnab tsho sab saum toj yog ntsuab. PDB cov lis dej num rau kev sib tshooj siv lead ua muaj xws li hauv qab no: 4LOH (cGAMP, ntsuab), 6UKV (MSA-02, daj), 6XNP (SR717, txiv kab ntxwv), 4LOL (DMXAA, magenta), thiab 4JC5 (CMA, liab). (b) Kev faib tawm ntawm cov amino acids tseem ceeb hauv cheeb tsam sab saum toj thiab hauv qab. Cov seem nyob rau hauv qab hnab tshos yog pom nyob rau hauv magenta, thiab cov ntsuab sawv daws yuav nyob rau hauv lub sab saum toj cheeb tsam.
Daim duab 5. Crystal complexes ntawm DMXAA thiab Merck 18. (a) Intermolecular contacts nyob rau hauv complex ntawm DMXAA thiab mSTING. Qhov khi DMXAA yog pom hauv cov xim grey, nrog rau ib tus neeg STING subunits nyob rau hauv lub symmetrical dimer pom ntsuab thiab ntshav. A thiab B superscripts qhia cov protein monomer lossis tus kheej ligand tus kheej. (b) Qhov nrug ntawm qhov tseem ceeb residues R237A thiab R237B, feem, thiab DMXAA (molecule A). R237A rau carboxyl ntawm DMXAA yog 6.02 Å, R237B rau carboxyl ntawm DMXAA yog 3.06 Å. (c) Cov qauv siv lead ua ntawm Merck 18 khi rau hSTING protein (PDB 6MXE) thiab cov ntsiab lus ntawm nws cov kev sib txuas sib txuas. Cov ligand khi tau pom hauv cov xim grey, nrog rau ib tus neeg STING subunits nyob rau hauv lub symmetrical dimer pom ntsuab thiab ntshav. A thiab B superscripts qhia cov protein monomer lossis tus kheej ligand tus kheej
Daim duab 6. Kev tshawb fawb hauv paus thiab peb cov hotspots optimized rau DMXAA thiab CMA derivatives. (a) Superposition ntawm tus qauv ntawm DMXAA khi rau mSTING (PDB: 4LOL, pom raws li tsaus xiav) nrog cov qauv ntawm DMXAA khi rau hSTING S162A / G230I / Q266I (PDB: 4QXR, pom li txiv kab ntxwv). (b) Cov ntsiab lus ntawm DMXAA khi rau mutated hSTING (PDB: 4QXR). Cov kab dashed kab sawv cev rau qhov chaw hloov kho uas muaj peev xwm. Amino residues qhia nyob rau hauv ntsuab yog mutated residues (S162A/Q266I).
Peb pom tias qhov tsis muaj peev xwm ntawm DMXAA los qhib txoj kev hosting yog cuam tshuam nrog qhov chaw ntawm DMXAA hauv qab hnab tshos (raws li Tshooj 2.1.1), uas kuj tau muab peb lub tswv yim tsim ntawm STING inhibitors: nce kev sib cuag ntawm DMXAA derivatives nrog rau thaj tsam hauv qab los ntawm kev hloov kho cov qauv kom STING nyob rau hauv ib qho kev tsis ua haujlwm. S162 thiab Q266 pw zoo nyob rau hauv qab ntawm qhov chaw sib khi, yog li DMXAAs tuaj yeem ua tau zoo nyob rau hauv kev sib cuag nrog ob qho tib si, ua kom yooj yim rau kev tshawb nrhiav cov nyhuv ntawm hauv qab hnab tshos amino acids ntawm ligands. Cov qauv-kev sib raug zoo kev sib raug zoo (SARs) kev tshuaj xyuas tau qhia tias 5, 6-dimethyl moiety thiab C7- txoj hauj lwm methoxy yog qhov tseem ceeb rau kev ua haujlwm lom neeg ntawm 3 thiab 4. Raws li pom hauv daim duab 6b, 5 ,6- pawg dimethyl tsim hydrophobic kev sib cuam tshuam nrog ntau cov amino acids hauv qab hnab tshos; vim qhov sib thooj ntawm C7 txoj hauj lwm ntawm DMXAA rau amino acid Q266, qhov sib txuas methoxy hloov pauv tau tso cai 3 thiab 4 los khi rau hosting. Yog li ntawd, peb cov txheej txheem elaborations presupposed lub locking ntawm 5, 6-dimethyl thiab C7 methoxy, thiab tus tsim ntawm tshiab derivatives tsom rau kev hloov kho pawg polar ntawm C1/C2 txoj hauj lwm ( cuam tshuam rau S162) thiab hloov cov carboxylic acid pawg ( ncua kev sib txawv ntawm cov qauv). Peb tsim thiab sib txuas ua ke ntawm cov derivatives siv cov tebchaw 3 thiab 4 raws li scaffolds, yog li siv tau rau peb qhov kev xav inhibitor conjecture.
2.2. Chemistry
Kev npaj ntawm txhua qhov nruab nrab thiab lub hom phiaj sib txuas tau piav qhia hauv Daim Ntawv Qhia 1. 1-methoxy-2, 3-dimethyl-4-nitrobenzene (5) thiab 2-bromobenzoic acid ( 7) tau muag khoom. Thaum pib, 1-methoxy-2, 3-dimethyl-4-nitrobenzene tau hloov mus rau 4-methoxy- 2,3-dimethylaniline (6) ntawm kev txo qis nrog hlau hmoov. Tom qab ntawd, 6 thiab 7 tau raug Ullmann cov tshuaj tiv thaiv nrog poov tshuaj carbonate nrog catalysis los ntawm tooj liab thiab tooj liab (I) oxide nyob rau hauv N, N-dimethylformamide los muab qhov sib thooj nruab nrab 8. Cov tshuaj tiv thaiv intramolecular condensation ntawm 8 tau ua nrog Eaton's reagent kom them nyiaj nruab nrab. 2-methoxy-3, 4-dimethylacridin-9(10H)-ib (9). Kev hloov pauv tom qab ntawm 9 tau ua nrog ethyl bromoacetate, thiab poob ntawm 1 equiv ntawm HBr afforded ethyl 2-(2-methoxy-3, 4-dimethyl{34}} oxoacridin -10(9H)- yl)acetate (10). Thaum kawg, ethyl acetate hauv 10 tau hydrolyzed rau carboxylic acid los ntawm sodium hydroxide yielding 2-(2-methoxy-3,4- dimethyl-9-oxoacridin-10 (9H)- yl) acetic acid (11).
Scheme 1. a. EtOH, Fe, NH4Cl; b. DMF, Cu, Cu2O, K2CO3; c. Eaton tus reagent; d. BrCH2COOC2H5, NaH; e. NaOH; f. CCl3CH(OH)2, NH2OH; g. H2SO4; h. EtOH, H2O2, NaOH.
Rau kev sib txuas tom qab ntawm ntau lub hom phiaj, peb yuav tsum xub ua cov khoom sib xyaw ua ke 2-amino-5-methoxy-3, 4-dimethylbenzoic acid (14). 6 tau reacted nrog chloral hydrate thiab hydroxylamine, tsim acetamide thiab hydroxyamino, nrog rau nruab nrab (E)-2-(hydroxylamine)-N-(4-methoxy-2,3-dimethylphenyl acetamide (12). Tom qab Beckmann rearrangement cov tshuaj tiv thaiv ntawm hydroxyamino ntawm 12 tau ua nrog sulfuric acid kom them taus lactam derivative 13. Thaum kawg, intermediate 13 yog hydrolyzed nrog hydrogen peroxide thiab sodium hydroxide nyob rau hauv ethanol tov affording 14. Siv 14 raws li cov khoom pib rau cov tshuaj tiv thaiv, peb. ntxiv ua ke ib series ntawm 8-methoxy-9, 10-dimethyl-6Hpyrrolo[3,2,1-de]acridine-1,6( 2H)-dione derivatives nrog hloov pauv ntawm R1 thiab R2. Thaum pib, 14 thiab 15-19 tau raug Ullmann cov tshuaj tiv thaiv nrog potassium carbonate nrog catalysis los ntawm tooj liab thiab tooj liab (I) oxide nyob rau hauv N, N-dimethylformamide los muab cov intermediates sib thooj 20-24. Cov tshuaj intramolecular condensation ntawm 20-24 tau ua nrog Eaton's reagent kom them taus 8-methoxy-9,10-dimethyl-6H-pyrrolo[3,2,{46} }de]acridine-1,6(2H)- dione derivatives (25–29). Ib yam li ntawd, siv 14 raws li cov khoom pib rau cov tshuaj tiv thaiv, peb ntxiv synthesized ib series ntawm 7-methoxy-5,6-dimethyl-9-oxo-9,{{ 59}}dihydroacridine-4- carboxylic acid derivatives nrog hloov pauv ntawm R. Thaum pib, 14 thiab 7 lossis 30 tau raug Ullmann cov tshuaj tiv thaiv nrog potassium carbonate nrog catalysis los ntawm tooj liab thiab tooj liab (I) oxide hauv N, N-dimethylformamide los muab Cov tshuaj intermediates 31 thiab 32. Cov tshuaj tiv thaiv intramolecular condensation ntawm 31 thiab 32 tau ua nrog Eaton's reagent kom them taus 7-methoxy-5, 6-dimethyl-9-oxo-9 , 10-dihydroacridine-4-carboxylic acid derivatives (33,34). Yog li, peb tsim, tsim, thiab tshuaj xyuas 16 acridone analogs, uas nws cov qauv tau sau tseg hauv Table 1.
Table 1. Cov qauv ntawm cov Analogs tshiab ntawm Txoj Kev Kawm Tam Sim No.
2.3. Binding Potencies ntawm Cov Khoom Tshiab rau STING thiab lawv SARs
Binding Potencies ntawm Cov Cheeb Tsam Tshiab rau STING thiab lawv cov SARs kom ncaj qha paub meej tias qhov ua tau zoo ntawm kev hloov kho cov qauv, peb thawj zaug tshuaj xyuas tag nrho cov khoom sib xyaw ua ke los ntawm cGAMP hloov chaw. Ntxiv nrog rau kev xaiv hom, muaj tsib qhov sib txawv ntawm STING uas yog polymorphic hauv tib neeg, R232 (WT, 58% ntawm cov pejxeem), HAQ (20%), H232 (13%), AQ (7%), thiab Q ( 2%) [32]. Yog li ntawd, peb siv cov khoom siv sib tw ua lag luam los ntawm homogeneous time-resolved fluorescence (HTRF) technology los ntsuam xyuas biochemical potency ntawm cov tebchaw tshiab ntawm mSTING thiab ntau yam hSTING isoforms (WT, H232, thiab AQ) thiab muab piv rau kev tswj. Raws li kev cia siab, kev tswj cov tebchaw 1–4 yog tag nrho cov khi rau mSTING; los ntawm hSTING npo, cov tebchaw 3 thiab 4 muaj IC50 qhov tseem ceeb ntawm qib micromolar.
Table 2. Cov txiaj ntsig ntawm cov tshuaj sib xyaw nrog ntau yam hSTING isoforms thiab mSTING kev sib tw binding kev soj ntsuam.
Ua ntej, raws li kev sib xyaw 3, halogen atom lossis methoxy pawg tau qhia ntawm C1 / C2 qhov chaw (Table 1). Compound 27, raws li qhov zoo tshaj plaws derivative ntawm compound 3 (C1 txoj hauj lwm hloov nrog F), muaj tag nrho cov kev ua ub no zoo dua li compound 3 (ib tug lej micromolar kev ua si nyob rau hauv tag nrho cov biochemical ntsuam xyuas, Table 2). Lub R1- hloov kho compound 25 muaj qhov dav spectrum binding nyhuv tab sis tsis ua haujlwm zoo li F-hloov lossis tsis hloov pauv. Muab piv rau compound 3, cov tebchaw (26, 28, thiab 29) nrog rau C2-site qhia pab pawg tag nrho pom qhov sib txawv ntawm kev txo qis hauv kev sib koom ua ke, tshwj xeeb tshaj yog cov compound 29. Thib ob, ua kom lub nraub qaum ntawm compound 4 thiab hloov lub halogen atom tawm tsam C1 / C2 qhov chaw, peb tau txais cov tebchaw 35–38 (Table 1). Cov tebchaw no tsis ua haujlwm nyob rau hauv tag nrho plaub qhov kev hloov pauv ntawm qhov siab txog li 100 µM, qhia tau tias kev hloov pauv nrog halogen atom ntawm C1 / C2 qhov chaw tsis raug zam. Tom qab ntawd, peb muab acetate pawg ntawm compound 4 rau hauv pawg carboxyl (33) thiab qhia methoxy ntawm C2 qhov chaw (34). Hmoov tsis zoo, qhov kev hloov pauv no yog qhov ua tsis tiav vim tias ob qho tib si tseem tsis muaj zog. Kawm los ntawm peb qhov ua tsis tiav, peb tau tshawb nrhiav ntxiv SARs ntawm cov khoom sib txuas 4 derivatives los ntawm kev hloov cov acetate pawg mus rau N txoj hauj lwm thiab tawm hauv C1 / C2 txoj haujlwm tsis hloov. Compound 11 qhia kev khi rau ntau yam ntawm STING variants, tag nrho nrog IC50s hauv qab 20 µM; qhov kev ua si yog superior rau compound 4 thiab piv rau compound 27. Tsis tas li ntawd, compound 9 (tsis muaj carboxyl pawg ntawm N site) thiab compound 10 (N-acetate ethyl) yog hluavtaws precursors ntawm compound 11 uas tsis tuaj yeem khi rau STING, indirectly qhia. qhov tseem ceeb ntawm pawg carboxylic acid.
2.4. Cellular Biological ntsuam xyuas
2.4.1. Hauv Vitro Kev Tshuaj Ntsuam Xyuas Tshiab Analogs nrog STING Agonist Kev Ua Si
Peb tau tshuaj xyuas txhua qhov sib xyaw ua ke rau STING agonist kev ua haujlwm siv 293T hSTING-WT, 293T-mSTING, thiab THP1-KO-STING cov xov tooj ntawm tes (muab los ntawm Invivo Gen). Siv 293T-hSTING-R232 hlwb thiab 293T-mSTING hlwb, peb tau pom kev ua haujlwm ntawm IRF txoj hauv kev raws li kev ntsuas tsis ncaj ntawm hom I IFN induction los ntawm kev saib xyuas cov haujlwm secretory embryonic alkaline phosphatase (SEAP), pab rau peb txoj kev tshawb fawb ntawm hom kev xaiv ntawm tshuaj sib xyaw. THP1-KO-STING hlwb tau tsim los ntawm THP1-Dual cells los ntawm kev ruaj khov knockout ntawm STING gene, thiab peb ua hauj lwm THP1-KO-STING hlwb kom paub meej tias cov compound puas muaj kev ua haujlwm ntawm STING-dependent cytokine induction. Peb piv cov STING agonist kev ua ub no ntawm tag nrho cov hom phiaj derivatives rau cov neeg siv agonists rau murine (DMXAA) thiab tib neeg (20, 30 -cGAMP) STING. Kuj ceeb tias, tsis muaj ib qho kev sib txuas ua ke tuaj yeem ua rau hSTING lossis mSTING txoj hauv kev (qhov siab tshaj plaws ntawm 200 µM), uas yog qhov tsis sib haum xeeb nrog cov txiaj ntsig ntawm kev txheeb xyuas. Yog li ntawd, peb cov STING binding agents uas tau soj ntsuam xyuas tej zaum yuav muaj peev xwm binding qauv raws li tshiab STING antagonists.
2.4.2. Hauv Vitro Kev Ntsuam Xyuas ntawm STING Binders nrog STING Inhibitor Cov Haujlwm
Ua ntej, peb tau ua qhov kev sim ua ntej: nrog rau 1 µM covalent inhibitor H151 raws li kev siv tau zoo, peb tau tshawb xyuas nws cov qib inhibitory hauv mSTING thiab hSTING cov xov tooj ntawm tes sib koom ua ke nrog ntau qhov sib txawv ntawm 20 3 0 -cGAMP los txiav txim siab qhov kev pom zoo. rau kev tshuaj xyuas tus inhibitor (saib Tshooj 4.2.3 rau cov lus qhia raws tu qauv). Qhov thib ob, peb pib soj ntsuam qhov inhibitory potency ntawm cov tebchaw tshiab los ntawm kev kho nrog cov kev sim ua ntej ntawm qhov concentration ntawm 100 µM. Peb pom tias qhov dav-spectrum STING-binding compounds 11 thiab 27 tau pom zoo inhibition nyob rau hauv ob qho tib si murine thiab tib neeg STING reporter hlwb, nrog cov tebchaw 3 thiab 4 thiab lwm cov tebchaw tsis ua haujlwm zoo. Qhov thib peb, peb teeb tsa cov concentration gradients rau cov tebchaw 11 thiab 27 los ntsuas ob qho tib si IC50 qhov tseem ceeb thiab sib piv nrog H151. Cov tshuaj 11 thiab 27 tau ua haujlwm ntawm micromolar concentrations inhibiting 20 3 0 -cGAMP-induced IFN- qhia hauv ob qho tib si hSTING thiab mSTING txoj hauv kev.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Nrog IC50 qhov tseem ceeb, compound 11 (hSTING 19.93 µM thiab mSTING 15.47 µM) outperformed compound 27 (hSTING 38.75 µM thiab mSTING 30.81 µM) (Daim duab 7a). Hauv kev sib piv, IC50 qhov tseem ceeb ntawm H-151 hauv 293T-hSTING thiab 293T-mSTING hlwb yog 1.04 thiab 0.82 µM (Daim duab 7a), qhia tau hais tias covalent inhibitor H-151 nthuav tawm cov nyhuv inhibitory zoo dua ntawm STING -dependent signaling nyob rau hauv kev soj ntsuam ntawm tes. Txhawm rau kom paub meej ntxiv txog cov cuab yeej inhibitory ntawm cov tebchaw 11 thiab 27, peb ua haujlwm lwm tus THP-1 human monocyte reporter cell kab THP1-Dual-hSTING-R232, uas siv dual-reporter system los qhia txog kev ua IRF raws li kev ntsuas tsis ncaj ntawm hom I IFN induction thiab ntawm NF-κB ua kom ua kom tsis muaj zog ntawm proinflammatory cytokine induction. Tom qab THP-1 xov xwm xov tooj ntawm tes stimulation nrog 20, 30 -cGAMP, peb ntxiv STING inhibitors nrog rau cov concentrations tsim nyog rau inhibiting inductions ntawm I IFN thiab proinflammatory cytokine. Raws li kev cia siab, cov tebchaw 11, 27, thiab H151 cuam tshuam STING-triggered IRF thiab NF-κB txoj kev ua haujlwm (Daim duab 7b). Tsis tas li ntawd, txhawm rau tshem tawm cov txiaj ntsig ntawm cytotoxicity ntawm cov tebchaw ntawm cov haujlwm inhibitory, peb siv CellTiter-Glo cov khoom siv los ntsuas kev ua haujlwm ntawm tes. Txaus siab rau, cov tebchaw 11 thiab 27 tsis pom muaj pov thawj ntawm cytotoxicity rau 293T thiab THP-1 hlwb los ntawm kev soj ntsuam cell viability thaum ntxiv ntawm cov concentrations (5 mus rau 100 µM) uas inhibit STING, sib piv rau H151, uas twb tau pom cytotoxicity tseem ceeb ntawm 10 µM (Daim duab 7c). Cov kev tshawb fawb no tau txheeb xyuas cov tebchaw 11 thiab 27 raws li qhov nruab nrab STING inhibitors, rhuav tshem hom rhiab heev thiab inhibiting IRF thiab NF-κB ob txoj hauv kev yam tsis muaj cytotoxicity.
2.5. Lub hauv paus ntawm Kev Ua Haujlwm ntawm Compound 11 tau tshawb xyuas los ntawm Docking
Cov txheej txheem ntawm kev sib tw STING antagonists yog los tuav qhov chaw sib txuas thiab cuam tshuam qhov kev ua kom zoo ntawm STING protein [20,21]. Tshwj xeeb rau hSTING, antagonists tawm ntawm hSTING dimer hauv qhov "qhib" conformation. Peb siv lub Glide docking los txiav txim qhov kev sib cuam tshuam ntawm tib neeg STING CTD (PDB ID code 6MXE) thiab qhov zoo tshaj plaws active compound 11. Nyob rau hauv lub docking qauv, ob lub tebchaw 11 yog ib nrab mus rau ib leeg thiab pw hauv qab ntawm lub cleft ntawm. lub hSTING dimer (Daim duab 8a), xauv hSTING nyob rau hauv ib tug inactive qhib conformation (Daim duab S2). Compound 11 feem ntau ua rau muaj kev sib cuam tshuam hydrophobic, nrog rau cov qauv tricyclic ntawm ligand tso cai rau kev sib cuag nrog lub interface hauv qab hnab tshos. Raws li nrog DMXAA los yog CMA, pawg carboxyl tsim ib daim ntawv cog lus hydrogen nrog sab saw ntawm T263, thaum pawg keto tsim ib daim ntawv cog lus hydrogen nrog cov saw ntawm T267. methoxy ntawm txoj hauj lwm C2 cuam tshuam nrog S162, ua rau ligand-ligand ib puag ncig ntawm ib leeg. Cov tshuaj bis-methyl tsis tsuas yog tsim cov ligand-ligand kev sib cuam tshuam tab sis kuj tseem nyob ntawm qhov chaw nkag mus rau lub hnab tshos sab saum toj, uas tiv thaiv kev khi ntawm ntuj STING agonists rau cov seem tseem ceeb (Daim duab 8b).
Daim duab 7. Broad-spectrum STING-binders 11 thiab 27 yog STING antagonists. (a) Cov khoom siv tshuaj lom neeg ntawm cov tebchaw 11 thiab 27, thiab cov haujlwm inhibitory ntawm STING inhibitors tawm tsam ob txoj hauv kev m- thiab h- STING. 293T hlwb (mSTING los yog hSTING), pretreated nrog sib txawv concentrations ntawm compounds tau stimulated los ntawm 20,30 -cGAMP; inhibition ntawm IRF txoj kev ua haujlwm tau ntsuas tsis ncaj los ntawm qhov ntsuas qhov muag (OD) qhov tseem ceeb. Qhov koob tshuaj-dependent inhibitory nkhaus tau haum los xam cov IC50s ntawm cov tebchaw. (b) Cov tshuaj 11 thiab 27 tuaj yeem cuam tshuam kev ua haujlwm ntawm hSTING dual txoj kev. THP1-hSTING-R232 cov xov tooj xov tooj ntawm tes tau txhawb nqa nrog 20,30 -cGAMP, thiab kho nrog cov tshuaj, inhibiting IFN- induction (xws li kev ua haujlwm los ntawm cov teeb meem txheeb ze (RLU) ntawm Lucia luciferase ) thiab proinflammatory cytokine induction (saib xyuas cov haujlwm ntawm SEAP ntsuas los ntawm OD cov nqi). (c) Cov analogs tshiab pom muaj cytotoxicity tsawg hauv kev sib piv nrog covalent inhibitor. 293T (los yog THP-1 hlwb) tau incubated nrog qhov qhia tau hais tias concentration ntawm inhibitors rau lub sij hawm qhia. Cell viability tau ntsuas los ntawm CellTiter-Glo cov khoom siv. Cov ntaub ntawv qhia yog qhov nruab nrab ntawm peb qhov kev sim ywj pheej. NS (tsis muaj qhov tseem ceeb) p> 0.05, *** p < 0.001. p tus nqi raug xam los ntawm ob-tailed t-test.
Daim duab 8. Docking qauv ntawm compound 11 khi rau STING protein. (a) Kev khi hom kev sib txuas 11. Cov ligand khi yog pom hauv cov xim grey, nrog rau ib tus neeg STING subunits nyob rau hauv lub symmetrical dimer pom ntsuab thiab ntshav. Hauv cov hnab ris khi ntawm STING monomer, hauv qab yog xim magenta thiab sab saum toj yog ntsuab. (b) Cov kev sib txuas sib txuas ntawm cov khoom sib txuas 11 yog khi rau STING. Compound 11 thiab hu rau STING amino acids yog qhia raws li tus qauv stick. Cov kev sib txuas ntawm cov tshuaj sib txuas thiab cov ntawv cog lus hydrogen tau pom nrog cov kab daj daj. A thiab B superscripts qhia cov protein monomer lossis tus kheej ligand tus kheej.
3. Kev sib tham
Raws li cov lus ceeb toom dav dav ntawm cov txheej txheem thiab cov qauv siv lead ua ntawm STING, peb muaj lub sijhawm los piav qhia txog qhov teeb meem kev tshawb fawb ntawm vim li cas DMXAA tsis muaj tib neeg STING agonistic ua haujlwm. Nyob rau hauv txoj kev tshawb no, los ntawm kev sib piv ntawm ntau yam STING siv lead ua qauv, peb tau txais ib co nthuav tshawb pom: 1. Lub STING activation conformation yog ruaj khov; 2. Muaj cov txheej txheem ua kom sib txawv rau hSTING thiab mSTING; 3. DMXAA thiab CMA yog nyob rau hauv qab ntawm qhov chaw khi qhov txawv ntawm lwm cov agonists. Muab cov txheej txheem sib txawv ntawm mSTING thiab hSTING, mSTING agonists DMXAA lossis CMA nyob hauv qab hnab tshos tsis tuaj yeem ua kom muaj zog hauv hSTING vim tias apo-hSTING muaj cov kab noj hniav loj dua tiv thaiv cov ligand los ntawm kev koom nrog R238.
Gao et al. qhia tias ib qho kev hloov pauv (G230I, S162A, thiab Q266I) tso cai hSTING nrog tib DMXAA rhiab heev li mSTING [33]. MD simulations tau qhia tias lub hau kev hloov pauv hauv cheeb tsam G230I sab saw yog txaus los tsim ib qho steric barrier los tiv thaiv DMXAA excretion, whereas DMXAA nkag tau tawm hauv hSTING WT [27]. Raws li kev hloov kho cov qauv sib txuas rau hSTING taw tes hloov pauv (S162A / Q266I) hauv qhov chaw sib txuas, DMXAA thiab CMA analogs muaj ntau tab sis tsis muaj hSTING agonists muaj zog. Yog vim li cas tom qab qhov no tej zaum yuav yog qhov sib txawv ntawm cov qauv kev sib txawv ntawm "natural" thiab "mutated" hSTINGs tuaj yeem ua lub luag haujlwm tseem ceeb hauv cov txheej txheem lees paub. Los ntawm MD simulations, Che et al. pom tias cov hSTINGs hloov pauv tsis zoo cuam tshuam cov kev sib koom tes ntawm cov dej molecules thiab hloov cov dej uas raug tshem tawm thaum ligand khi, uas yog qhov zoo dua rau kev rov ua kom DMXAA activation ntawm hSTING [31].
Txhawb los ntawm txoj kev tshawb nrhiav pom ntawm STING inhibitors nyob rau hauv qab khi qhov chaw [21], peb venture kom pom tias DMXAA, nyob rau hauv tib lub cheeb tsam, muaj peev xwm hloov mus rau tshiab STING inhibitors. Hauv kev tshawb fawb yav dhau los, peb tau sib xyaw cov qauv ntawm DMXAA thiab CMA nrog cov kev hloov pauv hloov pauv yooj yim thiab nrhiav tau zoo 3 thiab 4 uas tuaj yeem khi rau hSTING, tab sis lawv cov kev ua tsis muaj zog STING agonistic tsis phim lawv txoj kev sib khi potency. Hauv daim ntawv no, lub tswv yim tsim yog txhawm rau txhim kho kev sib cuag ntawm DMXAA analogs nrog rau hauv qab hnab tshos kom muaj kev sib tw tiv thaiv kev khi ntawm 20, 30 -cGAMP, yog li peb tsim thiab tsim cov qauv zoo ntawm cov khoom sib txuas 3 thiab 4.
cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob
Thawj kauj ruam ntawm kev tshuaj ntsuam yog los kuaj cov kev ua haujlwm biochemical siv cov khoom siv khi ntawm ntau yam STING variants los ua tus yam ntxwv ntawm kev khi lub zog ntawm cov khoom siv tshiab. Cov txiaj ntsig ntawm kev txheeb xyuas tau ua pov thawj zoo ntawm peb cov lus qhia tsim; peb tau txheeb xyuas 11 thiab 27 raws li broad-spectrum STING binders (zoo tshaj li 3 thiab 4) thiab tham txog SARs. Rau cov analogs ntawm compound 3, cov kev hloov kho ntawm C1 txoj hauj lwm zoo txhim kho txoj kev khi rau ntau yam STING variants, thaum C2- hloov cov tebchaw tsis muaj txiaj ntsig. Txawm li cas los xij, qhov sib xyaw 4 tsis tau txais txiaj ntsig zoo rau kev hloov kho cov qauv hauv C1 / C2. Tom qab hloov txoj haujlwm carboxylic acid (C4 rau N), peb pom qhov zoo dua STING binder 11 thiab txiav txim siab tias pawg carboxylic acid yog qhov tseem ceeb rau kev ua haujlwm. Tom ntej no, peb soj ntsuam tag nrho cov tebchaw rau cellular-theem kev ua si siv peb cov xov tooj ntawm tes, thiab tsis muaj lub hom phiaj tebchaw ua haujlwm. Tom qab ntawd peb tau tsim ib txoj kev tshuaj ntsuam rau STING inhibitors thiab rov tshuaj xyuas tag nrho cov analogs tshiab. STING binding agents 11 thiab 27 tau nthuav tawm micromolar inhibitory kev ua ub no, nrog cov ntaub ntawv piv rau kev sib tw binding assay. Covalent inhibitor H151 tau pom zoo me ntsis STING inhibitory kev ua haujlwm dua li 11 thiab 27 hauv vitro. Ntxiv rau, peb tau lees paub ntxiv tias 11 thiab 27 tuaj yeem cuam tshuam 20, 30 -cGAMP-induced activation ntawm STING dual txoj kev. Qhov zoo tshaj plaws, 11 thiab 27 tuav lub siab ntawm cell viability ntawm siab concentrations, uas yog tsis tau nrog H151. Cytotoxicity yog ib qho teeb meem tshwm sim nrog covalent inhibitors thiab txwv tsis pub siv H151.
Los ntawm kev txheeb xyuas docking, peb tau txais kev pom zoo rau hauv cov qauv kev ua haujlwm zoo tshaj plaws 11 kev ua haujlwm. Compound 11 nyob rau hauv qab hnab tshos zoo kawg nkaus, tuav hSTING nyob rau hauv ib tug inactive qhib conformation thiab yog li kev sib tw inhibiting 20, 30 -cGAMP khi. Cov qauv ntawm lub nplhaib tricyclic ntawm acridine yog qhov tseem ceeb los tsim kev sib cuam tshuam hydrophobic, nrog rau pawg carboxylic acid thiab ketone moiety anchoring lub compound nyob rau hauv qab ntawm lub hnab tshos. Peb kwv yees tias methoxy tsim kev sib raug zoo nrog S162, rub cov protein conformation kaw marginally tab sis tsis txaus los hloov nws mus rau hauv agonistic conformation. Cov qauv bis-methyl tsim ib qho chaw spatial-blocking uas txhim khu kev sib cuam tshuam ligand-ligand thiab detracts los ntawm agonist nkag mus rau hauv lub hnab tshos sab saum toj. Tam sim no, tsis muaj kev tshaj tawm cov qauv siv lead ua ntawm mSTING thiab STING antagonists, yog li peb tsis tuaj yeem piav qhia txog cov txheej txheem ntawm kev txiav txim ntawm 11 nrog mSTING siv txoj hauv kev docking. Txawm li cas los xij, peb kwv yees tias muaj ob txoj hauv kev: ib qho yog tias txoj cai ua yeeb yam zoo ib yam rau hSTING; lwm qhov yog qhov sib xyaw ua ke 11 khaws mSTING hauv qhov sib xyaw ua ke apo ntau dua thiab 20,30 -cGAMP tsis tuaj yeem nkag mus rau hauv hnab ris kom ua rau muaj kev cuam tshuam agonistic. Hauv cov ntsiab lus, peb qhov kev hloov kho hloov maj mam ntawm cov neeg tsis muaj zog STING agonists tau ua tiav qhov kev ua haujlwm lom neeg los nrhiav pom STING inhibitors. Cov kev tshawb pom no qhia txog qhov nyuaj ntawm STING cov hnab ris thiab muab cov kev tshawb fawb tshiab rau kev tsim tshuaj ntawm STING agonists lossis inhibitors.
4. Cov ntaub ntawv thiab cov txheej txheem
4.1. Chemistry
Cov kuab tshuaj thiab cov tshuaj reagents siv nyob rau hauv synthesis tau txais los ntawm Beijing Innochem Science thiab Technology Co., Ltd. (Beijing, Tuam Tshoj). Cov qauv ntawm cov khoom raug txheeb xyuas los ntawm 1H thiab 13C-NMR spectroscopy (JNM-ECA-400, Nyiv). Qhov hnyav molecular ntawm cov khoom tau ntsuas los ntawm high-resolution mass spectrometry (HRMS) nrog electrospray ionization (ESI) raws li hom ionization (Agilent 1260-G6230A, Lub teb chaws Yelemees). NMR thiab huab hwm coj spectra ntawm cov tebchaw muaj nyob rau hauv Cov Khoom Siv Ntxiv. Cheebtsam 35–38 tau tsim thiab txheeb xyuas los ntawm peb; thov xa mus rau cov lus tshaj tawm yav dhau los kom paub meej [23].
Ethyl {{0}}(2-methoxy-3,4-dimethyl-9-oxoacridin-10 (9H)-yl)acetate (1{ {25}}): Rau kev daws ntawm 4-methoxy{{10}}, 3-dimethylaniline (6) (0.97 g, 6.4 mmol) thiab 2-bromo-benzoic acid (7) (1.29 g, 6.4 mmol) hauv DMF (6 mL) ntawm chav tsev kub, peb ntxiv hmoov Cu (0.05 g), Cu2O ({{60}}}{182}}5 g) thiab K2CO3 ({{186}}}. Cov tshuaj tiv thaiv sib xyaw ua kom sov ntawm 110 ◦C rau 12 teev. Thaum tshem tawm cov kuab tshuaj nyob rau hauv lub tshuab nqus tsev, cov residue tau yaj hauv 1 N NaOH tov (25 mL). Cov khoom nyoos tau txais los ntawm nag lossis daus thaum acidification ntawm cov lim nrog conc. HCl. Tom qab ziab, cov khoom nyoos tau ntxiv rau Eaton's reagent (5 mL) ntawm chav tsev kub, tom qab ntawd cov sib tov tau muab rhaub rau 90 ◦C rau 1 h. Cov tshuaj tiv thaiv txias tau muab tso rau hauv cov kua dej saturated NaHCO3. Lub precipitate tau lim los sau cov khoom ntxhib. Purification ntawm residue los ntawm silica gel kem chromatography muab 9, ib tug daj ntseg daj khoom (0.61 g, 37.7% tawm los). Kev daws ntawm NaH (1.43 mmol) thiab 9 (0.33 g, 1.3 mmol) hauv DMF (5 mL) ntawm chav tsev kub yog nplawm rau 1 teev thiab tom qab ntawd txias rau 5-7 ◦C. Cov ethyl bromoacetate (0.43 g, 2.6 mmol) tau ntxiv rau qhov sib xyaw ua ke thiab txuas ntxiv stirred ntawm chav tsev kub rau 20 teev. Tom qab ua tiav cov tshuaj tiv thaiv (TLC), cov tshuaj tiv thaiv sib tov yog nchuav rau hauv dej khov (15 mL). Lub resulting precipitates tau lim tawm, qhuav, thiab ces muab rho tawm nrog chloroform. Evaporation ntawm cov kuab tshuaj muab ib tug crude ester uas yog purified los ntawm recrystallization muab 10, ib tug daj khoom (0.33 g, 76% tawm los). 1H NMR (400 MHz, DMSO-D6) δ 8.30 (d, J=8.4 Hz, 1H), 8.08 (d, J=8.6 Hz, 1H), 7.75–7.67 (m , 1H), 7.57–7.50 (m, 1H), 7.48 (s, 1H), 5.02 (s, 2H), 4.22 (q, J=7.1 Hz, 2H), 3.96 (s, 3H) , 2.77 (s, 3H), 2.34 (s, 3H), 1.21 (t, J=7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-D6) δ (ppm): 169.25, 157.87, 155.96, 147.46, 146.72, 135.46, 132.15, 130.17, 1218.511, 19.46, 95.26, 72.06, 61.28, 56.06, 14.59 TZS 14.34, 13.77 Nws. HRMS (ESI) m/z [M+H]+ xam rau C20H21NO4: 339.3910 pom: 340.1546. 2-(2-methoxy-3,4-dimethyl-9-oxoacridin-10(9H)-yl)acetic acid (11): Kev daws ntawm 10 (0.37 g, 1.1 mmol) thiab NaOH (0.05 g, 1.3 mmol) hauv ethanol (20 mL) thiab dej (2 mL) tau rhaub rau 60 ◦C rau 1 h. Tom qab tshem tawm ntawm cov kuab tshuaj, lub resulting residue yog yaj nyob rau hauv dej thiab lim. Tom qab neutralization ntawm filtrate nrog conc. HCl, qhov sib tov tau lim kom tau cov khoom nyoos, tom qab ntawd recrystallized los ntawm methanol muab 11, cov hmoov daj daj (0.29 g, 85.7% tawm los). 1H NMR (400 MHz, DMSO-D6) δ 10.51 (s, 1H), 8.33 (d, J=6.9 Hz, 1H), 8.04 (d, J=8.5 Hz, 1H ), 7.69 (s, 1H), 7.67–7.62 (m, 1H), 7.45 (t, J=6.8 Hz, 1H), 4.52 (s, 2H), 3.90 (s, 3H), 2.75 (s, 3H), 2.31 (s, 3H). 13C NMR (101 MHz, DMSO-D6) δ (ppm): 159.04, 155.63, 147.47, 146.64, 134.86, 131.52, 129.56, 128.98, 125.392.15. 01.77, 96.02, 74.81, 55.69, 14.42, 13.33 TZS . HRMS (ESI) m/z [M+H]+ xam rau C18H17NO4: 311.3370 pom: 312.1229.
{{0}}chloro-8-methoxy-9,10-dimethyl-6H-pyrrolo [3,2,1-de]acridine{ {9}},6(2H)-dione (25): Rau kev daws ntawm 6-carboxy-4-methoxy-2, 3-dimethylbenzenaminium (14) (1.3 g, 6.4 mmol) thiab 2-(2-bromo-4-chlorophenyl) acetic acid (15) (1.6 g, 6.4 mmol) hauv DMF (6 mL) ntawm chav tsev kub, peb ntxiv cov hmoov Cu. (0.05 g), Cu2O (0.05 g) thiab K2CO3 (0.71 g, 5.1 mmol). Cov tshuaj tiv thaiv sib tov tau rhaub ntawm 110 ◦C rau 12 teev. Thaum tshem tawm cov kuab tshuaj nyob rau hauv lub tshuab nqus tsev, cov residue tau yaj hauv 1 N NaOH tov (25 mL). Cov khoom nyoos tau txais los ntawm nag lossis daus thaum acidification ntawm cov lim nrog conc. HCl. Tom qab ziab, cov khoom nyoos tau ntxiv rau Eaton's reagent (5 mL) ntawm chav tsev kub, tom qab ntawd cov sib tov tau muab rhaub rau 90 ◦C rau 1 h. Cov tshuaj tiv thaiv txias tau muab tso rau hauv cov kua dej saturated NaHCO3. Lub precipitate tau lim los sau cov khoom ntxhib. Purification ntawm residue los ntawm silica gel kem chromatography muab 25, cov khoom daj (0.80 g, 38.6% tawm los). 1H NMR (400 MHz, DMSO-D6) δ 8.17 (d, J =8.1, 1.5 Hz, 1H), 7.86 (d, J=6.9 Hz, 1H), 7.51 (s, 1H), 3.84 (s, 3H), 3.78 (s, 2H), 2.49 (s, 3H), 2.27 (s, 3H); HRMS (ESI) m/z [M+H]+ xam rau C18H14ClNO3: 327.7640 pom: 328.0734. 4-chloro-8-methoxy-9, 10-dimethyl-6H-pyrrolo[3,2,{104}}de]acridine{105}} ,6(2H)-dione (26): Lub synthesis ntawm cov compound no zoo ib yam li 25. 15 tau hloov nrog 2-(2-bromo-5-chlorophenyl)acetic acid (16). Peb tau txais 0.82 g 26 raws li cov khoom daj-daj nrog cov txiaj ntsig ntawm 38.7%. 1H NMR (400 MHz, DMSO-D6) δ 7.73 (s, 1H), 7.52 (s, 1H), 7.38 (s, 1H), 3.71 (s, 3H), 3.64 (s, 2H), 2.36 (s, 3H), 2.13 (s, 3H); HRMS (ESI) m/z [M+H]+ xam rau C18H14ClNO3: 327.7640 pom: 328.0734.
{{0}}fluoro-8-methoxy-9,10-dimethyl-6H-pyrrolo[3,2,1-de]acridine{ {9}},6(2H)-dione (27): Lub synthesis ntawm cov compound no zoo ib yam li 25. 15 tau hloov nrog 2-(2-bromo-4-fluorophenyl)acetic acid (17). Peb tau txais cov khoom sib sib zog nqus daj 27 nrog 0.84 g (42.1% tawm los). 1H NMR (400 MHz, DMSO-D6) δ 8.21 (d, J=8.0 Hz, 1H), 7.68 (s, 1H), 7.23 (d, J {{39}) }.5 Hz, 1H), 3.88 (s, 3H), 3.81 (s, 2H), 2.53 (s, 3H), 2.30 (s, 3H); HRMS (ESI) m/z [M+H]+ xam rau C18H14FNO3: 311.3124 pom: 312.1030. 4-fluoro-8-methoxy-9, 10-dimethyl-6H-pyrrolo[3,2,1-}de]acridine-1 ,6(2H)-dione (28): Lub synthesis ntawm cov compound no zoo ib yam li 25. 15 tau hloov nrog 2-(2-bromo-5-fluorophenyl)acetic acid (18). 0.81 g tob daj khoom tau txais (40.5% tawm los). 1H NMR (400 MHz, DMSOD6) δ 7.86 (s, 1H), 7.64 (s, 1H), 7.51 (s, 1H), 3.83 (s, 3H), 3.77 (s, 2H), 2.49 (s, 3H), 2.26 (s, 3H); HRMS (ESI) m/z [M+H]+ xam rau C18H14FNO3: 311.3124 pom: 312.1030.
4,8-dimethoxy-9,10-dimethyl-6H-pyrrolo[3,2,1-de]acridine-1,6(2H )-dione (29): Lub synthesis ntawm cov compound no zoo ib yam li 25. 15 tau hloov nrog 2-({17}}bromo-5-methoxyphenyl)acetic acid (19). {{20}}.73 g daj khoom tau muab nrog 35.2% tawm los. 1H NMR (400 MHz, DMSO D6) δ 7.93 (s, 1H), 7.61 (s, 1H), 7.47 (s, 1H), 3.84 (s, 3H), 3.81 ( s, 3H), 3.67 (s, 2H), 2.44 (s, 3H), 2.28 (s, 3H); HRMS (ESI) m/z [M+H]+ xam rau C19H17NO4: 323.3480 pom: 324.1230. 7-methoxy-5,6-dimethyl-9-oxo-9,10-dihydroacridine-4-carboxylic acid (33): Rau kev daws ntawm 6-carboxy-4-methoxy-2, 3-dimethylbenzenaminium (14) (1.30 g, 6.4 mmol) thiab 2-bromo-benzoic acid (7) (1.29) g, 6.4 mmol) hauv DMF (6 mL) ntawm chav tsev kub, peb ntxiv hmoov Cu (0.05 g), Cu2O (0.05 g) thiab K2CO3 (0.71 g, 5.1 mmol). Cov tshuaj tiv thaiv sib tov tau rhaub ntawm 110 ◦C rau 12 teev. Thaum tshem tawm cov kuab tshuaj nyob rau hauv lub tshuab nqus tsev, cov residue tau yaj hauv 1 N NaOH tov (25 mL). Cov khoom nyoos tau txais los ntawm nag lossis daus thaum acidification ntawm cov lim nrog conc. HCl. Tom qab ziab, cov khoom nyoos tau ntxiv rau Eaton's reagent (5 mL) ntawm chav tsev kub, tom qab ntawd cov sib tov tau muab rhaub rau 90 ◦C rau 1 h. Cov tshuaj tiv thaiv txias tau muab tso rau hauv cov kua dej saturated NaHCO3. Lub precipitate tau lim los sau cov khoom ntxhib. Kev lim dej ntawm cov khoom seem los ntawm silica gel kem chromatography muab 33, cov khoom daj daj daj (1.00 g, 52.7% tawm los). 1H NMR (400 MHz, DMSO-D6) δ 13.00 (s, 1H), 10.28 (s, 1H), 8.01 (d, J=9.7 Hz, 1H), 7.69 (d , J=8.5 Hz, 1H), 7.48 (t, J=9.0 Hz, 1H), 7.34 (s, 1H), 3.67 (s, 3H), 2.32 (s, 3H) ), 2.10 (s, 3H); HRMS (ESI) m/z [M+H]+ xam rau C17H15NO4: 297.3100 pom: 298.1073. 2,7-dimethoxy-5,6-dimethyl-9-oxo-9,10-dihydroacridine-4-carboxylic acid (34): Cov synthesis ntawm cov compound no zoo ib yam li 33. Lub 7 tau hloov nrog 2-bromo{167}} methoxybenzoic acid (30). Cov khoom daj muab 1.05 g nrog 50.2% tawm los. 1H NMR (400 MHz, DMSO-D6) δ 12.96 (s, 1H), 10.45 (s, 1H), 7.84 (s, 1H), 7.54 (s, 1H), 7.50 (s, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 2.48 (s, 3H), 2.27 (s, 3H); HRMS (ESI) m/z [M+H]+ xam rau C18H17NO5: 327.3360 pom: 328.1179.
4.2. Kev Tshawb Fawb Txog Kev Nyuaj Siab
Mouse STING cov khoom siv khi, tib neeg STING WT cov khoom siv khi, tib neeg AQ STING cov khoom siv khi, thiab tib neeg H232 STING cov khoom siv tau yuav los ntawm Cisbio. 293T mSTING (ISG/KI-IFNb) hlwb, 293T hSTING-R232 (ISG/KI-IFNb) hlwb, THP1-KI-hSTING-R232 hlwb, THP1-KO-STING hlwb tau muas los ntawm Invivogen. 293T mSTING hlwb thiab 293T hSTING-R232 hlwb tau coj los siv DMEM (Gibco, Waltham, MA, USA), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA), 4.5 g / L qabzib (Sigma- Aldrich), 10% FBS (Gibco), Pen-Strep (100 U/mL-100 µM) (Gibco), 100 µM normocin (Invivogen, San Diego, CA, USA), thiab ntxiv nrog cov tshuaj tua kab mob xaiv (Invivogen blasticidin (10 µM), hygromycin (100 µM) thiab zeocin (100 µM). THP1-KO-STING hlwb thiab THP1-KI-hSTING-R232 hlwb raug coj los siv RPMI-1640 (Gibco), 2 mM L-glutamine (Sigma-Aldrich), 25 mM HEPES (Sigma-Aldrich), 10% heat-inactivated FBS (Gibco), PenStrep (100 U/mL-100 µM) (Gibco), 100 µM normocin (Invivogen), thiab ntxiv nrog cov tshuaj tua kab mob xaiv (Invivogen) blasticidin ( 10 µM) thiab zeocin (100 µM). QUANTI-Blue solution, QUANTI-Luc, 20, 30 -cGAMP tau yuav los ntawm Invivogen. H151 tau txais los ntawm Beijing Innochem Science thiab Technology Co., Ltd. (Beijing, Suav). CellTiter-Glo luminescent cell viability assay kit tau txais los ntawm Promega.
4.2.1. HTRF STING Binding Competitive Assay
Raws li cov lus qhia ntawm STING cov khoom siv khi, cov kev daws teeb meem hauv qab no tau txuas ntxiv mus rau txhua qhov dej hauv 384-zoo phaj: 5 µL ntawm cov khoom siv tshawb nrhiav los yog 2 0, 30 -cGAMP (zoo tswj ) nrog sib txawv concentrations los yog diluent (tsis zoo tswj); 5 µL ntawm tib neeg STING 6His-tagged protein (tsis zoo tswj qhov zoo tau ntxiv rau kev kuaj tsis pom); 10 µL ntawm STING ligand d2 thiab Anti 6His-Tb3+ premixed ua hauj lwm tov. Tom qab kaw lub phaj thiab incubation ntawm chav tsev kub rau 3 h, fluorescence qhov tseem ceeb ntawm 665 nm thiab 620 nm tau nyeem. Qhov piv ntawm ob qhov fluorescence intensities (665 nm / 620 nm) tau siv los kwv yees qhov kev sib txuas ntawm cov khoom sib txuas. IC50 (50% inhibitory concentration) qhov tseem ceeb tau suav nrog software GraphPad Prism.
4.2.2. Cellular Assay rau Kev Ntsuam Xyuas Cov Khoom Sib Txuas rau Kev Ua Haujlwm Agonistic
180 µL cov nqi ntawm 293T mSTING hlwb, 293T hSTING-R232 hlwb, thiab THP1-KOSTING hlwb raug tshem tawm tau muab faib rau hauv 96-zoo tiaj-hauv qab daim hlau nrog qhov ntom ntawm ~ 50,000 hlwb /well (293T) lossis ~100,{12}} cells/well (THP1). Ib qho nyiaj ntawm 20 µL ntawm saline los yog saline tov ntawm ib qho kev xeem compound, los yog 20, 30 -cGAMP raws li cov ntaub ntawv zoo tau ntxiv, thiab cov hlwb raug incubated ntawm 37 ◦C nrog 5% CO2 rau 48 h. Tom qab ntawd, ib qho nyiaj ntawm 20 µL ntawm supernatant tau ntxiv rau hauv 96-zoo tiaj tiaj-hauv qab phaj, ua raws li 180 µL ntawm QUANTI-Blue tov rau txhua qhov dej. Lub phaj tau incubated ntawm 37 ◦C rau 3 h, thiab SEAP theem (IRF txoj kev ua ub no) tau txiav txim siv lub spectrophotometer ntawm 620–655 nm.
4.2.3. Cellular Assay rau Kev Ntsuam Xyuas Cov Khoom Sib Txuas rau Kev Ua Phem Txhaum Cai
Kev sim ua ntej: 180 µL ntawm 293T mSTING hlwb, 293T hSTING-R232 hlwb (~50,000 hlwb/zoo) tau tshwm sim rau 48 h ntawm 37 ◦C hauv ib qho 5% CO2 incubator nrog 10 µL 20, 30 -cGAMP (3.125 µM, 6.25 µM) thiab 10 µL H151 (1.0 µM ). Tom qab ntawd, tus nqi ntawm 20 µL ntawm supernatant tau ntxiv rau hauv 96-zoo tiaj tiaj-hauv qab phaj, ua raws li 180 µL ntawm QUANTI-Blue tov rau txhua qhov dej. Lub phaj tau incubated ntawm 37 ◦C rau 3 h, thiab SEAP theem (IRF txoj kev ua ub no) tau txiav txim siv lub spectrophotometer ntawm 620–655 nm. 180 µL ntawm 293T mSTING hlwb, 293T hSTING-R232 hlwb raug tshem tawm tau muab faib rau hauv 96-zoo tiaj-hauv qab daim hlau nrog qhov ntom ntawm ~ 50,000 hlwb / qhov zoo. 10 µL 2 0, 30 -cGAMP (3.125 µM) thiab compound 11 lossis compound 27 (0.78 µM, 1.56 µM, 3.13 µM, 6.25 µM, 12.5 µM, 250 µM, µM) lossis H151 (0.08 µM, 0.16 µM, 0.31 µM, 0.63 µM, 1.25 µM, 2.5 µM, 5 µM) tau ntxiv, thiab cov hlwb raug muab tso rau ntawm 37 ◦C nrog 5% h. Tom qab ntawd, tus nqi ntawm 20 µL ntawm supernatant tau ntxiv rau hauv 96-zoo tiaj tiaj-hauv qab phaj, ua raws li 180 µL ntawm QUANTI-Blue tov rau txhua qhov dej. Lub phaj tau incubated ntawm 37 ◦C rau 3 h, thiab SEAP theem (IRF txoj kev ua ub no) tau txiav txim siv lub spectrophotometer ntawm 620–655 nm. IC50 qhov tseem ceeb tau suav los ntawm GraphPad software.
4.2.4 ib. Cellular Assay for Inhibition of STING Dual Pathways
180 µL ntawm THP1-KI-hSTING-R232 cell suspension tau muab faib rau hauv 96-zoo flatbottom daim hlau nrog qhov ntom ntawm ~ 100,000 cell/well. Ib qho nyiaj ntawm 10 µL 20, 30 -cGAMP thiab 10 µL compound 11 (20 µM) lossis compound 27 (33 µM) lossis H151 (2 µM) tau ntxiv, thiab cov hlwb raug tsim los ntawm 37 ◦C nrog 5% CO2 rau 24 h. Tom qab ntawd, tus nqi ntawm supernatant tau ntxiv rau hauv 96-zoo dawb (opaque) phaj, ua raws li QUANTI-Luc lossis QUANTI-Blue tov rau txhua qhov dej, thiab qib luminescence lossis SEAP qib tau nyeem raws li cov neeg tsim khoom cov lus qhia. Qhov no tso cai rau kev kawm ib txhij ntawm IFN regulatory factor (IRF) txoj hauv kev, los ntawm kev ntsuam xyuas cov haujlwm ntawm Lucia luciferase thiab NF-κB txoj hauv kev, los ntawm kev saib xyuas cov haujlwm ntawm SEAP.
4.2.5. CellTiter-Glo Luminescent Cell Viability Assay
Cell viability raug ntsuas los ntawm CellTiter-Glo luminescent cell viability assay kit raws li cov chaw tsim khoom cov lus qhia. Luv luv, cov hlwb raug incubated nrog peb qhov sib txawv (5 µM, 10 µM, 100 µM) ntawm compound 11 compound 27, lossis H-151 rau 48 h. 100 µL CellTiter-Glo reagent tau ntxiv rau hauv 96-zoo kuaj phaj rau 10 min, ces luminescence tau kaw.
4.3. Molecular Docking ntawm Compound 11
Cov qauv siv lead ua ntawm STING complex (PDB ID: 6MXE) tau coj los ntawm Protein Data Bank nkag thiab siv los ua qhov pib. Protein tau npaj siv Protein npaj ntawm Maestro thiab muab faib ua cov saw A thiab saw B. Peb tsim ib qho chaw khi hauv LBD ntawm STING monomer raws li cov ligand qub (Merck-18) siv Receptor Grid Generation. Peb siv SP precision ntawm Glide-docking rau lub molecular docking ib feem, tso cai rau compound 11 los tsim ntau tshaj 20 poses. Thaum kawg peb tau tsim 16 kev sib txuas ua ke (tag nrho hauv qab hnab tshos) thiab sim cov kev sib khi dawb zog ntawm cov complexes siv Prime MM-GBSA module hauv Schrödinger's software. Tsis tas li ntawd, raws li cov qhab nia docking, glide qauv qhab nia, thiab MMGBSA dG Bind qhab nia, peb xaiv qhov kev pom zoo uas tau qhab nia thawj hauv ob thiab qeb thib peb hauv ib qho (pom hauv Table S2). Thaum kawg, peb tau sib sau ua ke qhov zoo tshaj plaws ntawm cov saw hlau A thiab B kom tau txais cov qauv docking tiav.
Cov ntaub ntawv
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