CN117257955B - Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug - Google Patents
Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug Download PDFInfo
- Publication number
- CN117257955B CN117257955B CN202311534857.7A CN202311534857A CN117257955B CN 117257955 B CN117257955 B CN 117257955B CN 202311534857 A CN202311534857 A CN 202311534857A CN 117257955 B CN117257955 B CN 117257955B
- Authority
- CN
- China
- Prior art keywords
- gallium
- fpr
- ferredoxin reductase
- pseudomonas aeruginosa
- fprb
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000589517 Pseudomonas aeruginosa Species 0.000 title claims abstract description 73
- 102100036777 NADPH:adrenodoxin oxidoreductase, mitochondrial Human genes 0.000 title claims abstract description 71
- 101710192343 NADPH:adrenodoxin oxidoreductase, mitochondrial Proteins 0.000 title claims abstract description 71
- 101710104207 Probable NADPH:adrenodoxin oxidoreductase, mitochondrial Proteins 0.000 title claims abstract description 71
- 239000003814 drug Substances 0.000 title claims abstract description 38
- 229940079593 drug Drugs 0.000 title abstract description 27
- 229910052733 gallium Inorganic materials 0.000 claims abstract description 65
- GYHNNYVSQQEPJS-UHFFFAOYSA-N Gallium Chemical compound [Ga] GYHNNYVSQQEPJS-UHFFFAOYSA-N 0.000 claims abstract description 61
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 40
- 230000000694 effects Effects 0.000 claims abstract description 40
- 108010074122 Ferredoxins Proteins 0.000 claims abstract description 25
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 18
- 230000014509 gene expression Effects 0.000 claims abstract description 16
- 230000009467 reduction Effects 0.000 claims abstract description 10
- CHPZKNULDCNCBW-UHFFFAOYSA-N gallium nitrate Chemical compound [Ga+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O CHPZKNULDCNCBW-UHFFFAOYSA-N 0.000 claims description 52
- 108090000623 proteins and genes Proteins 0.000 claims description 50
- 150000003839 salts Chemical class 0.000 claims description 27
- 229940044658 gallium nitrate Drugs 0.000 claims description 26
- 239000000126 substance Substances 0.000 claims description 25
- 102000004169 proteins and genes Human genes 0.000 claims description 20
- 150000001875 compounds Chemical class 0.000 claims description 12
- -1 gallium ions Chemical class 0.000 claims description 11
- 230000005764 inhibitory process Effects 0.000 claims description 10
- 230000001603 reducing effect Effects 0.000 claims description 10
- 239000004599 antimicrobial Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- YEEGWNXDUZONAA-UHFFFAOYSA-K 5-hydroxy-2,8,9-trioxa-1-gallabicyclo[3.3.2]decane-3,7,10-trione Chemical compound [Ga+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YEEGWNXDUZONAA-UHFFFAOYSA-K 0.000 claims description 6
- KSFOVUSSGSKXFI-GAQDCDSVSA-N CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O Chemical compound CC1=C/2NC(\C=C3/N=C(/C=C4\N\C(=C/C5=N/C(=C\2)/C(C=C)=C5C)C(C=C)=C4C)C(C)=C3CCC(O)=O)=C1CCC(O)=O KSFOVUSSGSKXFI-GAQDCDSVSA-N 0.000 claims description 6
- UBQYURCVBFRUQT-UHFFFAOYSA-N N-benzoyl-Ferrioxamine B Chemical compound CC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCNC(=O)CCC(=O)N(O)CCCCCN UBQYURCVBFRUQT-UHFFFAOYSA-N 0.000 claims description 6
- 229960000958 deferoxamine Drugs 0.000 claims description 6
- UPWPDUACHOATKO-UHFFFAOYSA-K gallium trichloride Chemical compound Cl[Ga](Cl)Cl UPWPDUACHOATKO-UHFFFAOYSA-K 0.000 claims description 6
- 229950003776 protoporphyrin Drugs 0.000 claims description 6
- 239000004480 active ingredient Substances 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- ASYYOZSDALANRF-UHFFFAOYSA-K 3-bis[(2-methyl-4-oxopyran-3-yl)oxy]gallanyloxy-2-methylpyran-4-one Chemical compound [Ga+3].CC=1OC=CC(=O)C=1[O-].CC=1OC=CC(=O)C=1[O-].CC=1OC=CC(=O)C=1[O-] ASYYOZSDALANRF-UHFFFAOYSA-K 0.000 claims 2
- 229940126585 therapeutic drug Drugs 0.000 abstract description 13
- 230000001225 therapeutic effect Effects 0.000 abstract description 6
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 5
- 208000015181 infectious disease Diseases 0.000 abstract description 5
- 230000004060 metabolic process Effects 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000002458 infectious effect Effects 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 32
- 239000002773 nucleotide Substances 0.000 description 21
- 125000003729 nucleotide group Chemical group 0.000 description 21
- 238000007792 addition Methods 0.000 description 16
- 238000003197 gene knockdown Methods 0.000 description 16
- 108091027544 Subgenomic mRNA Proteins 0.000 description 14
- 239000006137 Luria-Bertani broth Substances 0.000 description 13
- 239000002253 acid Substances 0.000 description 12
- 101150026476 PAO1 gene Proteins 0.000 description 11
- 239000004098 Tetracycline Substances 0.000 description 10
- 229960002180 tetracycline Drugs 0.000 description 9
- XPCTZQVDEJYUGT-UHFFFAOYSA-N 3-hydroxy-2-methyl-4-pyrone Chemical compound CC=1OC=CC(=O)C=1O XPCTZQVDEJYUGT-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000002585 base Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 230000006698 induction Effects 0.000 description 7
- 238000012216 screening Methods 0.000 description 7
- 108091005804 Peptidases Proteins 0.000 description 6
- 239000004365 Protease Substances 0.000 description 6
- 150000007513 acids Chemical class 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 235000019364 tetracycline Nutrition 0.000 description 6
- 150000003522 tetracyclines Chemical class 0.000 description 6
- 238000010446 CRISPR interference Methods 0.000 description 5
- 102000005720 Glutathione transferase Human genes 0.000 description 5
- 108010070675 Glutathione transferase Proteins 0.000 description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 229930101283 tetracycline Natural products 0.000 description 5
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000006142 Luria-Bertani Agar Substances 0.000 description 4
- HYMLWHLQFGRFIY-UHFFFAOYSA-N Maltol Natural products CC1OC=CC(=O)C1=O HYMLWHLQFGRFIY-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 241000589516 Pseudomonas Species 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 229910052783 alkali metal Inorganic materials 0.000 description 4
- 150000001340 alkali metals Chemical class 0.000 description 4
- 150000001412 amines Chemical class 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- XZNUGFQTQHRASN-XQENGBIVSA-N apramycin Chemical compound O([C@H]1O[C@@H]2[C@H](O)[C@@H]([C@H](O[C@H]2C[C@H]1N)O[C@@H]1[C@@H]([C@@H](O)[C@H](N)[C@@H](CO)O1)O)NC)[C@@H]1[C@@H](N)C[C@@H](N)[C@H](O)[C@H]1O XZNUGFQTQHRASN-XQENGBIVSA-N 0.000 description 4
- 229950006334 apramycin Drugs 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 229960003722 doxycycline Drugs 0.000 description 4
- 229910001195 gallium oxide Inorganic materials 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 229940043353 maltol Drugs 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 108091033409 CRISPR Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108700039887 Essential Genes Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108010044467 Isoenzymes Proteins 0.000 description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 241000181693 Pseudomonas aeruginosa PA14 Species 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000001952 enzyme assay Methods 0.000 description 3
- 101150113295 fprB gene Proteins 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 3
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 3
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 2
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical compound OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- DPRMFUAMSRXGDE-UHFFFAOYSA-N ac1o530g Chemical compound NCCN.NCCN DPRMFUAMSRXGDE-UHFFFAOYSA-N 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- AEMOLEFTQBMNLQ-BKBMJHBISA-N alpha-D-galacturonic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-BKBMJHBISA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 125000000637 arginyl group Chemical class N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 2
- 159000000032 aromatic acids Chemical class 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- VDANGULDQQJODZ-UHFFFAOYSA-N chloroprocaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1Cl VDANGULDQQJODZ-UHFFFAOYSA-N 0.000 description 2
- 229960002023 chloroprocaine Drugs 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 150000001991 dicarboxylic acids Chemical class 0.000 description 2
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 2
- 229940043237 diethanolamine Drugs 0.000 description 2
- AJNVQOSZGJRYEI-UHFFFAOYSA-N digallium;oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[Ga+3].[Ga+3] AJNVQOSZGJRYEI-UHFFFAOYSA-N 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- CKHJYUSOUQDYEN-UHFFFAOYSA-N gallium(3+) Chemical compound [Ga+3] CKHJYUSOUQDYEN-UHFFFAOYSA-N 0.000 description 2
- 229940050410 gluconate Drugs 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 2
- 229940071870 hydroiodic acid Drugs 0.000 description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 2
- 229960003943 hypromellose Drugs 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 229910052749 magnesium Inorganic materials 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 150000007522 mineralic acids Chemical class 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- ACTNHJDHMQSOGL-UHFFFAOYSA-N n',n'-dibenzylethane-1,2-diamine Chemical compound C=1C=CC=CC=1CN(CCN)CC1=CC=CC=C1 ACTNHJDHMQSOGL-UHFFFAOYSA-N 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229940124531 pharmaceutical excipient Drugs 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229940069328 povidone Drugs 0.000 description 2
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 2
- 229960004919 procaine Drugs 0.000 description 2
- 230000008261 resistance mechanism Effects 0.000 description 2
- 238000007480 sanger sequencing Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 239000004925 Acrylic resin Substances 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 229920000881 Modified starch Polymers 0.000 description 1
- FBWADIKARMIWNM-UHFFFAOYSA-N N-3,5-dichloro-4-hydroxyphenyl-1,4-benzoquinone imine Chemical compound C1=C(Cl)C(O)=C(Cl)C=C1N=C1C=CC(=O)C=C1 FBWADIKARMIWNM-UHFFFAOYSA-N 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 208000032536 Pseudomonas Infections Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229940081735 acetylcellulose Drugs 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229940126575 aminoglycoside Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229940041011 carbapenems Drugs 0.000 description 1
- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
- 101150102092 ccdB gene Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- OIQPTROHQCGFEF-UHFFFAOYSA-L chembl1371409 Chemical compound [Na+].[Na+].OC1=CC=C2C=C(S([O-])(=O)=O)C=CC2=C1N=NC1=CC=C(S([O-])(=O)=O)C=C1 OIQPTROHQCGFEF-UHFFFAOYSA-L 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 108010035554 ferric citrate iron reductase Proteins 0.000 description 1
- 229910001448 ferrous ion Inorganic materials 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 229940124307 fluoroquinolone Drugs 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000012988 high-throughput synthesis Methods 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- CXKWCBBOMKCUKX-UHFFFAOYSA-M methylene blue Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 CXKWCBBOMKCUKX-UHFFFAOYSA-M 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 230000001937 non-anti-biotic effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 235000019645 odor Nutrition 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940040944 tetracyclines Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000004408 titanium dioxide Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 150000003952 β-lactams Chemical class 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/433—Thidiazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Inorganic Chemistry (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to the technical field of biological medicines, and provides an application of ferredoxin reductase Fpr as a target point of a pseudomonas aeruginosa therapeutic drug. The application of the ferredoxin reductase Fpr as the target point of the pseudomonas aeruginosa therapeutic drug provided by the application takes the ferredoxin reductase Fpr as the target point of the pseudomonas aeruginosa therapeutic drug, and inhibits the ferredoxin reductasefprThe gene expression and/or the reduction of the Fpr activity of the ferredoxin reductase can obviously improve the effectiveness of the gallium antibacterial agent on pseudomonas aeruginosa. Based on the above, the application takes the ferredoxin reductase Fpr as a target point of the anti-pseudomonas aeruginosa drug, combines a gallium antibacterial agent and an agent for inhibiting the expression or activity of the ferredoxin reductase Fpr, can block the key metabolic process of the survival and the infectious capacity of pseudomonas aeruginosa, and becomes a novel antibacterial and therapeutic scheme.
Description
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to application of ferredoxin reductase Fpr serving as a target point of a pseudomonas aeruginosa therapeutic drug.
Background
Pseudomonas aeruginosa, also known as Pseudomonas aeruginosaPseudomonasaeruginosa) Is ubiquitous in the environment, has flagella, and belongs to gram-negative bacteria. The bacteria are considered as conditional pathogens of pneumonia of people with low immunity such as cystic fibrosis or chronic obstructive pulmonary disease, and are also the main causes of morbidity and mortality of the patients; in addition, pseudomonas aeruginosa causes inflammation of the blood and urinary tract and tissue destruction. According to statistics, pseudomonas aeruginosa occupies the first place in non-zymotic bacteria infection, and is one of the most common pathogenic bacteria in nosocomial infection; the epidemiological monitoring data of pathogenic bacteria show that the infection in the Pseudomonas aeruginosa hospital is more serious.
In recent years, the clinical antibiotic therapy has been aggravated by the decreasing sensitivity of pseudomonas aeruginosa to drugs such as fluoroquinolones, aminoglycosides, carbapenems, and beta-lactams due to the excessive and irregular use of broad-spectrum antibiotics. Therefore, in addition to the need for an in-depth understanding of the pathogenic and drug-resistant mechanisms of pseudomonas aeruginosa, there is also a need to attempt to screen for novel, efficient, safe and less resistant antimicrobial agents from existing non-antibiotic agents.
Disclosure of Invention
The application aims to provide application of ferredoxin reductase Fpr as a target point of a pseudomonas aeruginosa therapeutic drug, and aims to solve the problem that the pseudomonas aeruginosa therapeutic effect is limited.
In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides the use of a ferredoxin reductase Fpr as a target for a therapeutic drug for pseudomonas aeruginosa.
In a second aspect, the present application provides a method of inhibiting ferredoxin reductasefprUse of a substance that expresses a gene and/or reduces the activity of a ferredoxin reductase Fpr for the preparation of a medicament for the treatment of pseudomonas aeruginosa, which inhibits ferredoxin reductasefprThe substance for expressing genes and/or reducing the activity of ferredoxin reductase Fpr comprises a compound shown in a formula I or pharmaceutically acceptable salt thereof,
formula I.
Further, the medicine, such as a medicine for treating or screening pseudomonas aeruginosa, also comprises gallium antibacterial agents.
Further, the gallium antibacterial agent includes at least one of gallium nitrate, gallium chloride, gallium maltol, gallium citrate, deferoxamine gallium, protoporphyrin gallium, and nano gallium oxide. It is understood that the gallium based antimicrobial agent is not limited to that shown above.
Further, the ferredoxin reductase Fpr includes at least one of ferredoxin reductase FprA and its cognate proteins, ferredoxin reductase FprB and its cognate proteins.
In a third aspect, the present application provides a combination for the treatment of pseudomonas aeruginosa comprising inhibiting ferredoxin reductasefprSubstances for gene expression or reduction of the activity of ferredoxin reductase Fpr, and gallium-based antibacterial agents, which inhibit ferredoxin reductasefprThe substance for gene expression or reducing the activity of ferredoxin reductase Fpr includes a compound represented by formula I or a pharmaceutically acceptable salt thereof,
formula I.
Further, the gallium antibacterial agent is at least one of gallium nitrate, gallium chloride, gallium maltol, gallium citrate, deferoxamine gallium, protoporphyrin gallium and nano gallium oxide.
Further, the combination also comprises pharmaceutically acceptable auxiliary materials.
The application of the ferredoxin reductase Fpr provided by the first aspect of the application as a target point of a pseudomonas aeruginosa therapeutic drug plays an important role in maintaining the growth of pseudomonas aeruginosa under the conditions of oxidization, permeation and metal stress pressure, so that the ferredoxin reductase Fpr is used as the target point of the pseudomonas aeruginosa resistant drug, and has a wide application prospect in the pseudomonas aeruginosa therapeutic drug.
The second aspect of the present application provides an inhibiting ferredoxin reductasefprUse of a substance that expresses a gene and/or reduces the activity of a ferredoxin reductase Fpr for the preparation of a medicament for the treatment of pseudomonas aeruginosa, due to inhibition of ferredoxin reductasefprGene expression and/or reduction of the Fpr activity of ferredoxin reductase can increase the sensitivity of gallium ion antibacterial agents to Pseudomonas aeruginosa, thereby effectively improving the therapeutic effect of the antibacterial agents and thus inhibiting ferredoxin reductasefprThe substance for expressing genes and/or reducing the activity of ferredoxin reductase Fpr has wide application prospect in medicines for resisting pseudomonas aeruginosa.
The combination provided in the third aspect of the present application comprises inhibiting ferredoxin reductasefprGene listSubstances that achieve or reduce the activity of ferredoxin reductase Fpr, and gallium-based antimicrobial agents. Experimental analysis shows that by inhibiting ferredoxin reductase in pseudomonas aeruginosafprThe gene expression or the reduction of the activity of ferredoxin reductase Fpr can obviously improve the effectiveness of the gallium antibacterial agent on pseudomonas aeruginosa. Based on the above, the combination of the two drugs can block the key metabolic process of the survival and infection ability of the pseudomonas aeruginosa, become a novel antibacterial and therapeutic scheme, and provide a novel thought and approach for the treatment of the pseudomonas aeruginosa.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following description will briefly introduce the drawings that are needed in the embodiments or the description of the prior art, it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the results of CRISPRi-seq screening for genes related to gallium stress provided in the examples of the present application;
FIG. 2 is a graph of gallium pressure provided in the examples of the present applicationfprBKnocking down a bacterial growth curve;
FIG. 3 is a graph of gallium pressure provided in the examples of the present applicationfprAKnocking down a bacterial growth curve;
FIG. 4 is a graph of gallium pressure provided in the examples of the present applicationfprBKnocking out the growth curve of the strain and the strain of the anaplerosis;
FIG. 5 is a gallium nitrate pair provided in an embodiment of the present applicationfprAAndfprBeffects after combined action;
FIG. 6 is an in vitro enzyme activity assay for FprB protein provided in the examples herein.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.
In this application, the term "and/or" describes an association relationship of an association object, which means that there may be three relationships, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
It should be understood that, in various embodiments of the present application, the sequence number of each process does not mean that the sequence of execution is sequential, and some or all of the steps may be executed in parallel or sequentially, where the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weights of the relevant components mentioned in the embodiments of the present application may refer not only to specific contents of the components, but also to the proportional relationship between the weights of the components, and thus, any ratio of the contents of the relevant components according to the embodiments of the present application may be enlarged or reduced within the scope disclosed in the embodiments of the present application. Specifically, the mass described in the specification of the embodiment of the present application may be a mass unit that is well known in the chemical industry field such as μ g, mg, g, kg.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
The term "Dox" is an abbreviation for "Doxycycline" and denotes a tetracycline.
The term "Fpr" is an abbreviation for "Ferredoxin NADP (+) reduction" and indicates Ferredoxin reductase.
The term "FprA" is "subs I Fpr" and denotes the ferredoxin reductase FprA.
The term "FprB" is "subs II Fpr" and denotes the ferredoxin reductase FprB.
Currently, overuse of antibiotics leads to the emergence of resistant bacteria, adding difficulty to the treatment of pseudomonas aeruginosa. Gallium based antimicrobial agents have attracted attention by their unique antimicrobial mechanisms. Gallium antibacterial agents are different from other antibiotics in that pharmacological properties of gallium depend on chemical mimicry, and it is difficult for bacteria to resist gallium by drug resistance mechanisms such as target mutation, drug modification or alternative metabolic pathways.
Based on the above, the embodiment of the application takes the ferredoxin reductase Fpr as the target point of the pseudomonas aeruginosa therapeutic drug by inhibiting the ferredoxin reductasefprThe gene expression and/or the reduction of the ferredoxin reductase Fpr can improve the effectiveness of the gallium antibacterial agent on the pseudomonas aeruginosa, further effectively improve the treatment effect of the gallium antibacterial agent on the multi-drug resistant pseudomonas aeruginosa, and provide a new thought and approach for the treatment of the pseudomonas aeruginosa.
Some embodiments of the present application provide for the use of a ferredoxin reductase Fpr as a target for a pseudomonas aeruginosa therapeutic drug.
Pseudomonas aeruginosa, an opportunistic human pathogen, can survive and reproduce in severe environments such as high temperature, high salt concentration and toxic chemicals, depending on its excellent pressure resistance. Among them, ferredoxin reductase Fpr plays an important role in maintaining bacterial growth under oxidative, osmotic and metallic stress pressures. Therefore, the ferredoxin reductase Fpr is used as a target point of the anti-pseudomonas aeruginosa drug, and has wide application prospect in preparation and/or screening of the pseudomonas aeruginosa therapeutic drug.
Some embodiments of the present application provide an inhibition of ferredoxin reductasefprApplication of substance for expressing genes and/or reducing activity of ferredoxin reductase Fpr in preparation of medicines for treating pseudomonas aeruginosa and inhibiting ferredoxin reductasefprThe substance for expressing genes and/or reducing the activity of ferredoxin reductase Fpr comprises a compound shown in a formula I or pharmaceutically acceptable salt thereof,
formula I.
Experimental analysis proves that the iron oxide-reducing enzyme Fpr is used as a target point of the pseudomonas aeruginosa therapeutic drug, and the action mechanism is as follows: by inhibition of ferredoxin reductasefprThe gene expression and/or the reduction of the Fpr activity of the ferredoxin reductase can obviously improve the effectiveness of the gallium antibacterial agent on the pseudomonas aeruginosa, thereby providing a new thought and approach for the treatment of the pseudomonas aeruginosa.
Experiments prove that the iron oxide protein reductase FprA is necessary for the growth of pseudomonas aeruginosa, is an essential gene of the pseudomonas aeruginosa, and is an effective antibacterial target. Thus, inhibition of ferredoxin reductasefprThe substance for expressing genes and/or reducing the activity of ferredoxin reductase Fpr has wide application prospect in medicines for resisting pseudomonas aeruginosa.
Of course, because the ferredoxin reductase Fpr is used as a target point of a pseudomonas aeruginosa treatment drug, the development of the drug related to pseudomonas aeruginosa treatment can be promoted, and therefore, the ferredoxin reductase Fpr is used as a target point, and the ferredoxin reductase Fpr has wide application prospect in preparing and screening anti-pseudomonas aeruginosa drugs.
It will be appreciated that the compound of formula I is designated 1- [5- (4-Chloro-benzoylsulfanyl) - [1,3,4] thiadiazzol-2-yl ] -3- (4-Chloro-phenyl) -urea by International Union of pure and applied chemistry (International Union of Pure and Applied Chemistry, IUPAC) nomenclature and has a molecular weight of 411.32.
The compound with the structural formula shown in the formula I or pharmaceutically acceptable salt thereof can effectively inhibit and reduce the activity of ferredoxin reductase Fpr, and has wide application prospect in the aspect of being used for treating multi-drug resistant pseudomonas aeruginosa by being combined with a gallium ion antibacterial agent.
It is understood that pharmaceutically acceptable salts include acid addition salts and base addition salts. Exemplary acid addition salts include, but are not limited to, salts from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphonic acid, salts from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids, and salts from amino acid-containing salts such as arginine salts, gluconate, galacturonate. Illustratively, the base addition salt is a complex of a base and an alkali metal or a base and an organic amine. Wherein the alkali metal includes, but is not limited to, sodium, potassium, magnesium, and calcium; organic amines include, but are not limited to, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1, 2-diamine), N-methylglucamine, and procaine.
In some embodiments, the medicament, such as a medicament for treating or screening pseudomonas aeruginosa, further comprises a gallium based antimicrobial agent. It is understood that the gallium-based antibacterial agent refers to an antibacterial agent containing gallium ions as a main active ingredient.
Experimental analysis proves that the ferredoxin reductase Fpr is used as a target point of the pseudomonas aeruginosa therapeutic drug by inhibiting the ferredoxin reductasefprThe gene expression and/or the reduction of the Fpr activity of the ferredoxin reductase can obviously improve the effectiveness of the gallium antibacterial agent on pseudomonas aeruginosa. Based on the above, the application takes the ferredoxin reductase Fpr as the target point of the anti-pseudomonas aeruginosa drug, combines the gallium antibacterial agent and the substance for inhibiting the expression or the activity of the ferredoxin reductase Fpr, and can block the survival and the activity of pseudomonas aeruginosaThe key metabolic process of infectious ability provides a novel antibacterial and therapeutic regimen. Meanwhile, pharmacological properties of the gallium antibacterial agent depend on chemical mimicry, and drug resistance mechanisms such as target mutation, drug modification or alternative metabolic pathways are difficult to enable bacteria to resist gallium, so that gallium-containing preparations such as the gallium antibacterial agent are used as potential non-traditional antibacterial agents, and the treatment effect of multi-drug resistant bacteria pseudomonas aeruginosa infection can be effectively improved.
In some embodiments, the gallium-based antimicrobial agent includes, but is not limited to, at least one of gallium nitrate, gallium chloride, gallium maltol, gallium citrate, deferoxamine gallium, protoporphyrin gallium, and nano-gallium oxide. The gallium antibacterial agent and inhibiting ferredoxin reductasefprThe combination of gene expression and/or substances that reduce the activity of ferredoxin reductase Fpr can significantly improve the therapeutic effect on multi-drug resistant pseudomonas aeruginosa.
In some embodiments, the ferredoxin reductase Fpr comprises at least one of ferredoxin reductase FprA and its cognate, ferredoxin reductase FprB and its cognate. It will be appreciated that the ferredoxin reductase Fpr also includes at least one of the homologous proteins/isozymes of the ferredoxin reductase FprA, and of course also includes at least one of the homologous proteins/isozymes of the ferredoxin reductase FprB. Since there is a certain error in the amino acid sequences of the ferredoxin reductase FprA and the ferredoxin reductase FprB, and their homologous proteins and isozymes in different strains of pseudomonas aeruginosa, a protease homologous to ferredoxin reductase FprA, a protease homologous to ferredoxin reductase FprB are also included in the scope of the ferredoxin reductase Fpr (see table 1, where PDB is a protein database Protein Data Bank abbreviation). It will be understood by those skilled in the art that homologous proteases refer to proteases that have similar amino acid sequences, similar structures, and similar functions.
TABLE 1 homologous proteases of FprA and FprB
Pseudomonas aeruginosa exists with two genes encoding ferredoxin reductase Fpr, respectivelyfprAGene and genefprBAnd (3) a gene.fprBThe gene has iron reductase activity and promotes the Fenton reaction when Pseudomonas aeruginosa is treated with antibiotics that can cause oxidative stress.fprAGenes play an important role in the defense of bacteria against oxidative stress and ferrous ion depletion. And our data displayfprAIs an essential gene of pseudomonas aeruginosa and is important for the growth of the bacillus. Thereby, inhibition offprAGene and genefprBThe expression of the gene or the reduction of the activity of FprA/FprB becomes a new strategy for treating the pseudomonas aeruginosa, and has important significance for the treatment of the pseudomonas aeruginosa.
Some embodiments of the present application provide a combination for the treatment of pseudomonas aeruginosa comprising inhibiting ferredoxin reductasefprA substance that expresses a gene and/or reduces the activity of ferredoxin reductase Fpr, and a gallium-based antibacterial agent. Experimental analysis of the examples of the present application demonstrates inhibition of ferredoxin reductasefprThe substance for expressing genes and/or reducing the activity of the ferredoxin reductase Fpr can obviously improve the bactericidal activity of the gallium antibacterial agent on pseudomonas aeruginosa, and based on the substance, the gallium antibacterial agent is used together with a drug for inhibiting the expression or the activity of the ferredoxin reductase Fpr, so that the treatment effect of the pseudomonas aeruginosa, in particular to multiple drug resistant pseudomonas aeruginosa can be obviously improved.
It is understood that the gallium-based antibacterial agent refers to an antibacterial agent containing gallium ions as a main active ingredient.
In some embodiments, the inhibiting ferredoxin reductasefprThe substance for expressing gene and/or reducing the activity of ferredoxin reductase Fpr comprises a compound shown in a formula I or a pharmaceutically acceptable compound thereofThe salt is used as a salt,
formula I.
It is understood that pharmaceutically acceptable salts include acid addition salts and base addition salts. Exemplary acid addition salts include, but are not limited to, salts from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphonic acid, salts from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids, and salts from amino acid-containing salts such as arginine salts, gluconate, galacturonate. Illustratively, the base addition salt is a complex of a base and an alkali metal or a base and an organic amine. Wherein the alkali metal includes, but is not limited to, sodium, potassium, magnesium, and calcium; organic amines include, but are not limited to, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1, 2-diamine), N-methylglucamine, and procaine.
In some embodiments, the gallium-based antimicrobial agent includes, but is not limited to, at least one of gallium nitrate, gallium chloride, gallium maltol, gallium citrate, deferoxamine gallium, protoporphyrin gallium, and nano-gallium oxide. The gallium antibacterial agent and inhibiting ferredoxin reductasefprThe combination of gene expression and/or a substance that reduces the activity of ferredoxin reductase Fpr can significantly enhance the therapeutic effect of pseudomonas aeruginosa.
In some embodiments, the combination further comprises a pharmaceutically acceptable adjuvant.
Pharmaceutically acceptable pharmaceutical excipients are added into the medicine, so that the preparation and clinical application of the prepared medicine are ensured. In some embodiments, the pharmaceutical excipients are selected from at least one of diluents, wetting agents, binders, lubricants, colorants, coating agents. In some embodiments, the weight and volume of the medicament are enhanced primarily by the addition of diluents to facilitate shaping and dosing, in preferred embodiments of the invention the diluents are selected from, but not limited to, at least one of starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose. In some embodiments, the material can be moistened to create a viscosity of sufficient strength to facilitate granulation by the addition of a humectant, which in preferred embodiments of the present invention is selected from, but is not limited to, at least one of water, ethanol, glycerin. In some embodiments, the non-tacky or less tacky material is agglomerated and bound into particles by the addition of a binder, which in preferred embodiments of the present invention is selected from, but not limited to, at least one of Hypromellose (HPMC), povidone (PVP), starch slurry, syrup. In some embodiments, the coating agent and coloring agent are added to improve tablet appearance, increase drug stability, mask bad drug odors, and change the appearance of the particles; in a preferred embodiment of the present invention, the coating agent is selected from at least one of, but not limited to, acrylic resin, hypromellose, povidone, cellulose acetate; the colorant is selected from, but not limited to, at least one of titanium dioxide, sunset yellow, methylene blue.
In some embodiments, the dosage form of the combination for treating pseudomonas aeruginosa is selected from at least one of tablets, capsules, granules, pills, injections, suspensions, dispersants, syrups. However, the formulation for treating pseudomonas aeruginosa is not limited thereto, and other formulation can be realized within the scope of the present invention.
The following description is made with reference to specific embodiments.
Materials and methods
1. Inducible multi-tetracycline-CRISPRi systems and library construction
1.1 an inducible multicycline (Doxycycline) -CRISPRi system was developed in pseudomonas aeruginosa PA 14. The construction flow of the system is as follows:
first, PA14 was subjected to whole genome re-sequencing. Extracting PA14 genome DNA, detecting the purity and the integrity of the DNA by agarose gel electrophoresis, randomly breaking into fragments with the length of about 350bp by an ultrasonic breaker, then adopting NEB Next Ultra ™ DNA Library Prep Kit for Illumina kit, and finishing the whole library preparation by the steps of terminal repair, A tail addition, sequencing joint addition, purification, PCR amplification and the like, and finally sequencing by Illumina NovaSeq PE150,150.
1.2 construction of CRISPRi cloning vectors comprisingdCas9、ccdB、sgRNA scaffold、R6K、Tn7L、Tn7RAnd codon optimizedTetRNamed pCRISPRi-TetR-dCAs9-ccdB. Wherein,ccdBthe gene expresses a toxic protein, and as a reverse screening mark, bsaI enzyme cutting sites are arranged at two ends, and bacteria successfully cloned by sgRNA after One-step golden gate can survive on LB solid culture.
1.3sgRNA design, based on the R language pipeline, the target whole genome sgRNA was designed based on the Pseudomonas aeruginosa PA14 genome sequencing results, with 2 sgRNAs per gene. After high-throughput synthesis of the sgRNA primers, the gene modules carrying different sgRNAs were inserted into the PA14 genome by means of ligation transfer and transposition insertion, using one-step gold gate method to clone onto the sgRNA cloning vector. At this time, each bacterial individual carries only one sgRNA targeting a particular gene in its genome.
2. CRISPRi-Seq screening of key genes responsive to gallium stress
After 14 generations of the constructed CRISPRi library were cultured in LB broth medium containing tetracyclines, the culture was continued with M9 medium with or without 200. Mu.M gallium nitrate for 21 generations. Bacteria were collected and genomic DNA was extracted and a single PCR was performed to prepare Illumina sequencing library. By comparing the abundance of the target specific gene sgRNA, genes sensitive to gallium nitrate after knockdown are identified. The results are shown in fig. 1, and as can be seen from fig. 1,FprBthe sensitivity of gallium to pseudomonas aeruginosa can be increased after gene knockout.
fprBFunctional and phenotypic verification of (2)
3.1fprBKnock-down strain construction: firstly, constructing donor strain which is engineering bacteriaE. coliWM3064, carry knock-downfprBWherein the sgRNA sequence comprises OXL2385 as shown in SEQ ID NO.1 and OXL2386 as shown in SEQ ID NO. 2.
OXL2385（SEQ ID NO.1）：5’-TATAGGTGAATTTCTCTTCGCTGG-3’，
OXL2386（SEQ ID NO.2）：5’-AAACCCAGCGAAGAGAAATTCACC-3’。
The auxiliary strain is engineering bacterium carrying plasmid for expressing integraseE. coliWM3064 (helper strain contains Tn7 transposon helper Plasmid, addgene: plasmid # 141161). The donor strain is pseudomonas aeruginosa PA14; single colonies of donor, recipient and helper strains were inoculated, respectively, into LB broth medium containing the corresponding antibiotics (helper strain: 300. Mu.M DAP+100. Mu.g/ml ampicillin, somatic strain: 300. Mu.M DAP+30. Mu.g/ml apramycin, recipient strain: no antibiotics) and cultured overnight at 37 ℃. Mu.l of each broth was centrifuged at 7000g for 2 min, the supernatant was discarded, and 1 ml of LB broth was added for resuspension and centrifugation, and repeated twice. The helper strain, donor strain and recipient strain were combined in a 1:1:1 volume ratio of about 300. Mu.l, 700. Mu.l LB broth was supplemented to 1 ml,7000g was centrifuged for 2 min, the supernatant was discarded, resuspended in 50. Mu.l LB broth, and incubated on LB agar plates containing DAP for 6-8 hours at 37 ℃. Scraping the bacterial plaque, re-suspending in 1 ml PBS, centrifuging 7000 Xg for 2 min, discarding supernatant, and repeating the steps for 2 times; the suspension was resuspended in 1 ml PBS and 20. Mu.l of the suspension was plated on LB agar plates containing 30. Mu.g/ml apramycin and incubated at 37℃for 14-16 hours. Colonies were verified by PCR and Sanger sequencing, and primers required for colony PCR included OXL299 with nucleotide sequence shown in SEQ ID No.3 and OXL with nucleotide sequence shown in SEQ ID No. 4:
OXL299（SEQ ID NO.3）：5’-GTTGCAATTGCTCTCGTACCATGTCG-3’，
OXL300（SEQ ID NO.4）：5’-CCACCCTGTGGGTATTGGGCATCG-3’。
the required sanger sequencing primer comprises OXL659 (unidirectional) with a nucleotide sequence shown in SEQ ID NO. 5:
OXL659（SEQ ID NO.5）：5’-TGCGACTACTCTTGCCTACTACCTA-3’。
3.2fprAknock-down strain construction: by sequence alignment analysis, found in Pseudomonas aeruginosafprAGene and genefprBThe amino acid sequence of the gene is 42% identical, and the simulated spatial structure has high overlapping degree. Wherein,fprAand (3) withFprBThe amino acid sequence of (2) is shown below.
fprAAmino acid sequence of (a):
MSNLYTERVLSVHHWNDTLFSFKTTRNPGLRFKTGQFVMIGLEVDGRPLMRAYSIASPNYEEHLEFFSIKVPDGPLTSRLQHLKEGDELMVSRKPTGTLVHDDLLPGKHLYLLSTGTGMAPFLSVIQDPETYERYEKVILVHGVRWVSELAYADFITKVLPEHEYFGDQVKEKLIYYPLVTREPFRNQGRQTDLMRSGKLFEDIGLPPMNPQDDRAMICGSPSMLEETSAVLDSFGLKISPRMGEPGDYLIERAFVEK。
fprBamino acid sequence of (a):
MTASEEKFTRQTLLDVQPLTPNLFTLRTSRDAGFRFRAGQFARLGVYKPSGSIVWRAYSMVSAPHDEFLDFFSIVVPDGEFTSELSRLREGDQLLVDRQAFGFLTLDRFVDGRDLWLLATGTGVAPFVSILQDFEVWERFESIKLVYSVRESKELAYRELIAGLAEREYLAEHAHKLQFIPVVTREQVPGCLNGRITTLIENGDLERAADLELTPEHSRVMLCGNPQMIEDTRAVLKARGMNLSLTRRPGQVAVENYW。
thus at the same time constructfprAThe strain was knocked down. Method 3.1, targetfprAComprises OXL2881 with nucleotide sequence shown as SEQ ID NO.6 and OXL2882 with nucleotide sequence shown as SEQ ID NO. 7:
OXL2881：5’-AGTGGTCTCATATAGTGCACGCTGAGAACGCGCTGTTTTGAGACCCTG-3’，
OXL2882：5’-CAGGGTCTCAAAACAGCGCGTTCTCAGCGTGCACTATATGAGACCACT-3’。
cloning of sgRNA onto pCRISPRi-TetR-dCAS9-ccdB vector and transformation intoE.coliWM3064 donor bacteria containing pCRISPRi-TetR-dCAS9-FprA sgRNA were obtained. Insertion into the genome of recipient bacteria by means of triparental binding and mini-Tn7 transpositionglmsDownstream. The obtained clone bacteria are verified by PCR and sequencing, and the primers are 3.1 identical.
3.3fprBKnock-out strain (Δf)prB) And anaplerotic strain (fprB-Com) construction: method for performing CRISPR/Cas9 technology in pseudomonas aeruginosa another important mode strain PAO1fprBKnockout of the gene.
The required sgRNA sequences include OXL2385 with the nucleotide sequence shown in SEQ ID NO.8 and OXL2386 with the nucleotide sequence shown in SEQ ID NO. 9:
OXL2385：5’-TATAGGTGAATTTCTCTTCGCTGG-3’，
OXL2386：5’-AAACCCAGCGAAGAGAAATTCACC-3’。
fprBextension station of gene upstream and downstream fragmentsThe desired primers are [ upstream primer OXL2387 (nucleotide sequence shown as SEQ ID NO. 10) and OXL2388 (nucleotide sequence shown as SEQ ID NO. 11) and downstream primer OXL2389 (nucleotide sequence shown as SEQ ID NO. 12) and OXL2390 (nucleotide sequence shown as SEQ ID NO. 13)]：
OXL2387：5’-GTCTTCCGTATCCAGCCGTCGTC-3’，
OXL2388：5’-GGTCCGCTCCGGATACCGATAC-3’，
OXL2389：5’-GTATCCGGAGCGGACCGTGCATGCCCGCGCAGTAGC-3’，
OXL2390：5’-CCGAAGGCTGCATTAGACTGCCTTTAC-3’。
Homologous repair templates (amplification primers OXL2387 and OXL 2390) were obtained after amplification of the upstream and downstream fragments by overlap PCR. Plasmids carrying Cas9 and sgrnas, and repair templates were electrotransferred to PAO1, screened on LB agar plates of tetracycline and carbenicillin sodium, and the resulting clones were PCR verified and Sanger sequenced, with the required primers including OXL2422 (nucleotide sequence shown in SEQ ID No. 14) and OXL2423 (nucleotide sequence shown in SEQ ID No. 15):
OXL2422：5’-CAAGACCCGACGCAAACGTATG-3’，
OXL2423：5’-TGGCCTACTGGAACAACGGCATG-3’。
clones that were verified to be correct were plasmid-deleted and maintained on LB agar plates containing 10% sucrose.
At the position offprBImplementation in knockout StrainfprBIs supplemented by (2)fprBExpression was induced by tetracycline. The pUC18T-mini-Tn7T-Gm vector is taken as a framework to supplement plasmid, a multi-tetracycline induction system pCon-TetR-pTet part is cloned to pUC18T-mini-Tn7T-Gm in an in-fusion mode, the obtained vector is pUC18T-mini-Tn 7T-Tet-Gm, required primers comprise OXL657 (the nucleotide sequence is shown as SEQ ID NO. 16), OXL658 (the nucleotide sequence is shown as SEQ ID NO. 17), OXL659 (the nucleotide sequence is shown as SEQ ID NO. 18) and OXL (the nucleotide sequence is shown as SEQ ID NO. 19):
OXL657：5’-GCAAGAGTAGTCGCAAAAAAATTAGCGCAAGAAGACAAAAATC-3’，
OXL658：5’-CCTGCTGATGTGCTCTGTGCTCAGTATCTCTATCACTGATAGGG-3’，
OXL659：5’-TGCGACTACTCTTGCCTACTACCTA-3’，
OXL660：5’-GAGCACATCAGCAGGTTTCACACAG-3’。
subsequent amplification from PAO1 genomefprBCoding sequence fragments, the required primers include OXL2472 (nucleotide sequence shown as SEQ ID NO. 20) and OXL2473 (nucleotide sequence shown as SEQ ID NO. 21):
OXL2472: 5’-CAGGAGGTACAATCAATGACCGCCAGCGAAGAGAAATTCA-3’，
OXL2473: 5’-GGCGCGAGGCAGGTGCTACCAGTAGTTCTCCACCG-3’。
after being spliced with pUC18T-mini-Tn 7T-Tet-Gm vector, the vector is transferred into WM3064 as donor bacteria, and is inserted into genome of acceptor bacteria by means of triparental binding and mini-Tn7 transpositionglmsDownstream. The obtained clone bacteria are verified by PCR and sequencing, and the primers are 3.1 identical. Primers required for amplifying pUC18T-mini-Tn 7T-Tet-Gm vector include OXL262 (nucleotide sequence shown as SEQ ID NO. 22) and OXL2027 (nucleotide sequence shown as SEQ ID NO. 23):
OXL262: 5’-CACCTGCCTCGCGCCGCAAAC-3’，
OXL2027: 5’-TGATTGTACCTCCTGGTCAGTGC-3’。
at the position offprBKnock-out strain (PAO 1 delta)fprB) Is performed in the middle offprAIs delta by PAO1 #)fprBAs recipient bacteria, triparental conjugation was performed with helper bacteria containing Tn7 transposon helper plasmid, and donor bacteria containing pCRISPRi-TetR-dCAS9-fprA sgRNA to obtain PAO1 deltafprBglmspCRISPRi-TetR-dCAs9-fprA, i.e., deltafprB-fprAkd; meanwhile, constructing a control mutant strain, and constructing PAO1 by taking PAO1 as a receptor bacterium and adopting a triparental conjugation modeglms::pCRISPRi-TetR-dCas9-fprAI.e. PAO1-fprAkd. The knockdown strains all have apramycin resistance genes and can grow in LB medium containing 30 mug/ml apramycin.
3.4 Phenotype verification:
will befprAAndfprBthe knockdown strain was inoculated in LB broth medium containing 30. Mu.g/ml apramycinThe medium was incubated until the absorbance OD600 was about 0.6, diluted 100-fold with fresh LB broth containing no or Dox, 50. Mu.l of the broth was inoculated into a transparent 96-well, 50. Mu.l of LB broth containing gallium nitrate at different concentrations was added to each well, the absorbance OD600 was continuously monitored in a Tecan Spark microplate reader after mixing, readings were taken every 10 minutes, and the results were continuously measured for 24 hours, as shown in FIGS. 2 and 3. Fig. 2 as a whole consists of A, B, wherein: a is a control bacterium, namely CRISPRi knockdown without targets is carried out in a wild strain, any gene on a genome is not knocked down after the induction of the doxycycline Dox, and the sensitivity to gallium nitrate with different concentrations is not influenced by the Dox; b isfprBThe sensitivity of the knockdown bacteria to gallium nitrate is increased after Dox induction. Fig. 3 as a whole consists of A, B, C, wherein: a isfprAKnock-down bacteria, which inhibit growth after different concentrations of Dox induction, have Dox concentration dependence, indicate thatfprAIs an essential gene; b is the influence of gallium nitrate with different concentrations on the growth of the gallium nitrate without Dox induction; c is the effect of different concentrations of gallium nitrate on growth after Dox induction of 6.25 ng/ml.
As can be seen from fig. 2 and 3, pseudomonas aeruginosa is more sensitive to gallium nitrate and appears to be growth-limited after addition of the tetracycline Dox; with the increase of the concentration of gallium nitrate, the effect of inhibiting pseudomonas aeruginosa is more obvious.
To evaluate gallium nitrate against wild-type PAO1 (PAO 1 wt),fprBKnock out (fprB) And anaplerotic strain (fprBCom), three strains were inoculated into LB broth medium, cultured until the absorbance OD600 was about 0.6, diluted 100-fold, 50 μl was inoculated into transparent 96 wells, and 50 μl of LB broth containing no or 50 μm gallium nitrate (final concentration 25 μm) was added to each well. Since the anaplerotic strain was multi-tetracycline Dox-induced, the medium was set to be free of and to be added with 12.5. 12.5 ng/ml multi-tetracycline Dox, absorbance OD600 was continuously monitored in a Tecan Spark microplate reader after mixing, readings were taken every 10 minutes, and the results were continuously measured for 24 hours as shown in FIG. 4. In fig. 4: PAO1 wt is wild type PAO1 deltafprBIs thatfprB knockout strainfprB-Com isfprBThe strain is knocked out and is a complement strain of the strain, and the complement strain is a tetracycline Dox inducible strain, delta strainfprBCom-no Dox is the Dox-free inducer, deltafprBCom-with Dox was the Dox-induced group, concentration 25 ng/ml. Fig. 4 as a whole consists of A, B, wherein: a is a gallium nitrate-free group and B is a 25. Mu.M gallium nitrate treated group. As can be seen from FIG. 4, 25. Mu.M gallium nitrate significantly inhibited the delta of the knockout strainfprBIs a growth of (a). Anaplerotic plant syndromefprBCom without doxycycline Dox induction, the trend of growth is markedly inferior to that of wild-type PAO1; after addition of Dox (25 ng/ml), the growth was significantly improved, approaching that of the wild-type strain.
To judgefprAAndfprBin the case of combined action, in the wild-type PAO1 (PAO 1 wt) andfprBknockout strain (fprB) Is made in (3)fprAKnocking down to obtain PAO1-fprAkd and DeltafprB-fprAkd strain. Respectively inoculating into LB broth culture medium, culturing until absorbance OD600 is about 0.6, diluting 100 times with fresh broth containing no or 25 ng/ml of multi-tetracycline Dox (final concentration is 12.5 ng/ml), inoculating 50 μl into transparent 96-well, and adding LB broth containing different concentrations of gallium nitrate (final concentrations are 3.13 μM, 6.25 μM, 12.5 μM and 25 μM) into each well. After mixing, absorbance OD600 was continuously monitored in a Tecan Spark microplate reader, readings were taken every 10 minutes, and the results were continuously measured for 24 hours, as shown in FIG. 5. Fig. 5 as a whole consists of A, B, C, D, wherein: a is PAO1-fprAkd bacterium (wild type PAO 1)fprAKnock down bacteria), does not contain Dox; b is PAO1-fprAkd bacterium (wild type PAO 1)fprAKnocking down bacteria), and performing Dox treatment; c is%fprB-fprAkd bacterium [ ]fprBAfter knockdownfprAKnock down bacteria), does not contain Dox; d is deltafprB-fprAkd bacterium [ ]fprBAfter knockdownfprAKnockdown bacteria), and subjected to Dox treatment. As can be seen from fig. 5, infprBIn the case of knockdown, knockdownfprAThe post pseudomonas aeruginosa tolerance concentration to gallium nitrate is 3.13 mu M from 6.25 mu M, and the tolerance capacity is weaker, namely the sensitivity to pseudomonas aeruginosa is higher.
FprB protein purification and enzyme activity assay
Will befprBCloning after amplification of the coding sequenceInto pGood_6p vector and transformed intoE. coliIn BL21 Rosetta, the expressed protein has a Glutathione S-transferase (GST) tag. The monoclonal was selected, cultured overnight at 37℃and 220rpm, the bacterial solution was 100-fold diluted into LB broth of 200 ml, the culture was continued until OD600 was 0.8, 1 mM IPTG (isopropyl. Beta. -D-thiogalactoside) was added, and the culture was carried out overnight at 16℃and 220rpm, thereby inducing the expression of FprB. The cells were collected by centrifugation at 5000 Xg for 10 minutes at 4 ℃. After resuspension of the pellet with 50ml buffer (containing 20 mM Tris-HCl,150 mM NaCl,pH =8.0), sonication was performed (power 400W, 2 seconds on, 4 seconds off, working time 20 minutes). The cell lysates were centrifuged at 10000 Xg for 45 min at 4 ℃. The supernatant was then collected and protein purified using GST beads, and the supernatant was incubated with GST beads at 4℃for 4 hours and centrifuged at 500 Xg for 10 minutes at 4 ℃. The non-specific binding was removed by elution with buffer (20 mM Tris-HCl,1M NaCl, pH=8.0). The protein was then eluted with elution buffer (containing 20 mM Tris-HCl,150 mM NaCl,20 mM GSSH). The addition of P3c protease removes the GST tag, so the final purified protein is unlabeled. Finally, the FprB eluate was further purified by molecular sieve, and the protein was stored in buffer (20 mM Tris-HCl,150 mM NaCl,pH =8.0, 20% glycerol) after elution. The FprB protein purified sample is collected after passing through a molecular sieve, and is frozen after collection and used for subsequent enzyme activity detection.
In vitro enzyme activity assay of FprB protein: a reaction solution containing 800 nM FprB, 400. Mu.M NADPH, 800. Mu.M DCPIP, 2% dimethyl sulfoxide (DMSO) and 50 mM Tris/HCl buffer (pH 8.0), together with gallium nitrate at different concentrations and compounds of formula I (concentrations set to 0, 3.13, 6.25, 12.5, 25, 50, 100 and 200. Mu.M) was added to a transparent 96-well microplate, 100. Mu.L total, and the absorbance at 620 nM was read in a Tecan Infinite M100PRO microplate reader, and the temperature was set to 25 ℃. The negative wells were reactions without FprB, with 3 parallel wells for each condition. The results are shown in FIG. 6. In fig. 6: a is the influence of gallium nitrate with different concentrations on the activity of FprB protein, and B is the influence of a compound shown in a formula I with different concentrations on the activity of FprB protein; in fig. 6, negative control was not supplemented with FprB. As can be seen from fig. 6, both gallium nitrate and the compound shown in formula I can inhibit the activity of FprB protein, and have concentration-dependent changes, and the inhibition effect is more obvious with the increase of the concentration of both.
The foregoing description of the preferred embodiments of the present application is not intended to be limiting, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the present application.
Claims (6)
1. Inhibition of ferredoxin reductasefprUse of a substance which expresses a gene and/or reduces the activity of a ferredoxin reductase Fpr for the preparation of a medicament for the treatment of pseudomonas aeruginosa, characterized in that the inhibition of ferredoxin reductasefprThe substance for expressing genes and/or reducing the activity of the ferredoxin reductase Fpr is a compound shown in a formula I or pharmaceutically acceptable salt thereof, and the medicine also comprises a gallium antibacterial agent, wherein the gallium antibacterial agent refers to an antibacterial agent taking gallium ions as main active ingredients;
formula I.
2. The use of claim 1, wherein the gallium-based antimicrobial agent comprises at least one of gallium nitrate, gallium chloride, gallium maltolate, gallium citrate, gallium deferoxamine, gallium protoporphyrin.
3. The use according to any one of claims 1 to 2, wherein the ferredoxin reductase Fpr comprises at least one of ferredoxin reductase FprA and its cognate proteins, ferredoxin reductase FprB and its cognate proteins.
4. A combination for the treatment of pseudomonas aeruginosa, comprising inhibition of ferredoxin reductasefprSubstances for gene expression or reduction of the activity of ferredoxin reductase Fpr, and gallium-based antibacterial agents, said substances inhibiting ferredoxin reductasefprThe substance for expressing genes or reducing the activity of the ferredoxin reductase Fpr is a compound shown in a formula I or pharmaceutically acceptable salt thereof, and the gallium antibacterial agent refers to an antibacterial agent taking gallium ions as main active ingredients;
formula I.
5. The combination of claim 4, wherein the gallium-based antimicrobial agent comprises at least one of gallium nitrate, gallium chloride, gallium maltolate, gallium citrate, gallium deferoxamine, gallium protoporphyrin.
6. The combination of any one of claims 4-5, further comprising pharmaceutically acceptable excipients.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311534857.7A CN117257955B (en) | 2023-11-17 | 2023-11-17 | Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311534857.7A CN117257955B (en) | 2023-11-17 | 2023-11-17 | Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117257955A CN117257955A (en) | 2023-12-22 |
| CN117257955B true CN117257955B (en) | 2024-03-15 |
Family
ID=89209041
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311534857.7A Active CN117257955B (en) | 2023-11-17 | 2023-11-17 | Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117257955B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5997912A (en) * | 1996-09-03 | 1999-12-07 | University Of Iowa Research Foundation | Method for inhibiting growth of P. aeruginosa using gallium-containing compounds |
| CN104473962A (en) * | 2005-11-01 | 2015-04-01 | 西奈山医学院(美国) | Growth control of oral and superficial microorganisms using gallium compounds |
| KR20150083466A (en) * | 2014-01-09 | 2015-07-20 | 연세대학교 산학협력단 | Inhibition of Elastase from Pseudomonas aeruginosa |
| CN108084088A (en) * | 2017-12-28 | 2018-05-29 | 华南农业大学 | A kind of quinolines JH62 in Pseudomonas aeruginosa source and its application |
-
2023
- 2023-11-17 CN CN202311534857.7A patent/CN117257955B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5997912A (en) * | 1996-09-03 | 1999-12-07 | University Of Iowa Research Foundation | Method for inhibiting growth of P. aeruginosa using gallium-containing compounds |
| CN104473962A (en) * | 2005-11-01 | 2015-04-01 | 西奈山医学院(美国) | Growth control of oral and superficial microorganisms using gallium compounds |
| KR20150083466A (en) * | 2014-01-09 | 2015-07-20 | 연세대학교 산학협력단 | Inhibition of Elastase from Pseudomonas aeruginosa |
| CN108084088A (en) * | 2017-12-28 | 2018-05-29 | 华南农业大学 | A kind of quinolines JH62 in Pseudomonas aeruginosa source and its application |
Non-Patent Citations (2)
| Title |
|---|
| CHARACTERIZATION OF HEME OXYGENASE AND FERREDOXIN NADPH REDUCTASE: TWO REDOX PARTNER PROTEINS INVOLVED IN THE HEME-IRON ACQUISITION PATHWAY OF PSEUDOMONAS AERUGINOSA;An Wang;《University of Kansas 学位论文》;正文第109-231页 * |
| Identification of Inhibitors Targeting Ferredoxin-NADP+ Reductase from the Xanthomonas citri subsp. citri Phytopathogenic Bacteria;Marta Martínez-Júlvez，等;《Molecules》;第23卷;摘要和正文第2-10页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117257955A (en) | 2023-12-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI778025B (en) | Antibiotic testing and screening system | |
| JP2020525012A5 (en) | ||
| WO2010079858A1 (en) | Novel bacteria-specific lysine proteins with broad antibacterial activity | |
| JP2011518543A (en) | Modified bacteriophages as adjuvants for antimicrobial agents and compositions and methods of use thereof | |
| Shuwen et al. | Intestinal phages interact with bacteria and are involved in human diseases | |
| US20200147129A1 (en) | Method, Target and Application for NO Accumulation to Decrease Pseudomonas Aeruginosa Invasiveness | |
| De | Mobile genetic elements of Vibrio cholerae and the evolution of its antimicrobial resistance | |
| CN112089824A (en) | Pharmaceutical composition containing polymyxin and application thereof | |
| CN117257955B (en) | Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug | |
| JP2022536068A (en) | Klevicin for control of Klebsiella | |
| US20230126514A1 (en) | Genetically engineered microorganisms that overexpress microcin-mge and methods of purification and use | |
| US20220265731A1 (en) | Modified escherichia coli strain nissle and treatment of gastrointestinal disorder | |
| Trevizol et al. | Resistance to polymyxins in Escherichia coli | |
| Ferguson | A study of clinical strains of Pseudomonas aeruginosa and the investigation of antibiotic resistance mechanisms in the multidrug resistant strain PA13 | |
| Bourgeois | Mining Toxic Proteins of Vibriophage ICP3 and Investigation of Type IE CRISPR/Cas Targeting in Vibrio cholerae | |
| WO2024030896A2 (en) | Bacteriophage compositions and methods for treatment of bacterial infections | |
| Modesto | Micro and macroevolution of multidrug resistant Klebsiella pneumoniae plasmids | |
| Alozie-Chidi | Why Do Some Human Associated Escherichia coli Strains Lack Antibiotic Resistance? | |
| de Sousa | Micro and Macroevolution of Multidrug Resistant Klebsiella Pneumoniae Plasmids | |
| US20140073639A1 (en) | Inhibition of antimicrobial targets with reduced potential for resistance | |
| Fodor et al. | An Overview of Multi-Antibiotic Resistance in Pathogenic Bacteria-From Selected Genetic and Evolutionary Aspects-A Review | |
| WO2024003301A1 (en) | Targeting e coli cells | |
| Peters | Identification and Characterization of Six Bacteriophages Capable of Infecting Extremely Antibiotic Resistant Strains of Stenotrophomonas maltophilia | |
| Nordraak | Antibiotics against bovine mastitis pathogens: resistance and novel targets | |
| Jonsdottir | Evolutionary trajectories of conjugative resistance plasmids and their interplay in the ecology of clinically relevant bacteria |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |