*Page 1* *Page 1*Reactive dirty fragments: implications for tuberculosis Reaktif fragmenkotor: implikasi untuk tuberkulosisdrug discovery penemuan obatPooja Gopal and Thomas Dick Pooja Gopal dan Thomas DickReactive multi-target fragments, old synthetic Reaktif fragmenmulti-target, sintetik tuaantimycobacterials that are activated inside Mycobacteriumantimycobacterials yang diaktifkan di dalam Mycobacteriumtuberculosis bacilli and are smaller than the usual drug-like, TB basildan lebih kecil dari yang biasa seperti obat,single-target molecules, represent critical components of molekul-targettunggal, merupakan komponen penting daricurrent tuberculosis chemotherapies. kemoterapi tuberkulosis saat ini.Recent studies showed Studi terbaru menunjukkanthat para-aminosalicylic acid is recognized as a substrate by bahwa asampara-aminosalisilat diakui sebagai substrat olehdihydropteroate synthase and poisons the downstream folate synthasedihidropteroat dan racun yang folat hilirpathway. jalur. Pyrazinamide, a key relapse-reducing drug, isPirazinamid, kambuh-mengurangi obat kunci, adalahmetabolized by an amidase and the reaction product interferesdimetabolisme oleh amidase dan ikut campur produk reaksiwith trans-translation, membrane potential and other targets. dengantrans-terjemahan, potensial membran dan target lainnya.However, the mechanism of action of pyrazinamide remains ill- Namun,mekanisme kerja pirazinamid tetap sewenang-wenangdefined and needs to be understood to rationally approach didefinisikandan perlu dipahami untuk secara rasional pendekatantreatment shortening. shortening pengobatan. The success of small dirtydrugs and Keberhasilan obat kecil kotor danprodrugs suggests that fragment-based whole cell screens prodrugsmenunjukkan bahwa layar seluruh sel berbasis fragmenshould be re-introduced in our current antimycobacterial drug haruskembali diperkenalkan dalam obat antimycobacterial kita saat inidiscovery efforts. upaya penemuan.Addresses AlamatAntibacterial Drug Discovery Laboratory, Department of Microbiology,Antibakteri Laboratorium Penemuan Obat, Departemen Mikrobiologi,Yong Loo Lin School of Medicine, National University Health System, YongLoo Lin School of Medicine, University Health System Nasional,National University of Singapore, 5 Science Drive 2, Block MD4A,National University of Singapore, 5 Science Drive 2, Blok MD4A,Singapore 117597, Republic of Singapore Singapura 117597, RepublikSingapuraCorresponding author: Dick, Thomas ( Sesuai penulis: Dick, Thomas (thomas_dick@nuhs.edu.sgthomas_dick@nuhs.edu.sg) )Current Opinion in Microbiology 2014, 21:712 Opini Lancar Mikrobiologi2014, 21: 7-12This review comes from a themed issue on Antimicrobials Ulasan iniberasal dari masalah bertema pada AntimikrobaEdited by James J Collins and Roy Kishony Diedit oleh James J Collinsdan Roy Kishonyhttp://dx.doi.org/10.1016/j.mib.2014.06.015http://dx.doi.org/10.1016/j.mib.2014.06.0151369-5274/# 2014 The Authors. 1369-5274 / # 2014 Penulis. Published byElsevier Ltd. This is an Diterbitkan oleh Elsevier Ltd Ini adalahopen access article under the CC BY-NC-ND license ( terbuka artikelakses di bawah CC BY-NC-ND lisensi (http://creative-http: // kreatif-commons.org/licenses/by-nc-nd/3.0/). commons.org/licenses/by-nc-nd/3.0/).Current TB chemotherapies and drug Kemoterapi TB sekarang dan obatdiscovery approaches pendekatan penemuanMycobacterium tuberculosis remains the most deadly bac- Mycobacteriumtuberculosis tetap bac- paling mematikanterial pathogen globally [ 1 ]. terial patogen secara global [ 1 ]. Tuberculosis (TB) is treated Tuberkulosis (TB) diperlakukanwith a combination therapy consisting of isoniazid (INH), dengan terapikombinasi yang terdiri dari isoniazid (INH),rifampicin (RIF), pyrazinamide (PZA), and ethambutol rifampisin (RIF),pirazinamid (PZA), dan etambutolfor 2 months, followed by 4 months of treatment with selama 2 bulan,diikuti oleh 4 bulan pengobatan denganINH and RIF. INH dan RIF. This lengthy regimen has of course Rejimenpanjang ini memiliki tentu sajaimplementation and compliance issues, and this in turn implementasi danmasalah kepatuhan, dan ini pada gilirannyafuels development of drug resistant disease [ 2,3 pengembanganbahan bakar penyakit resisten obat [ 2,3 ]. ]. Multi- Multidrug resistant TB, defined as being resistant to INH and TB yangresistan terhadap obat, yang didefinisikan sebagai resisten terhadap INHdanRIF, is treated with less potent and more toxic second line RIF,diperlakukan dengan baris kedua kurang kuat dan lebih beracundrugs, fluoroquinolones and injectables (aminoglycosides obat,fluoroquinolones dan injeksi (aminoglikosidaand capreomycin), and requires at least 2 years of therapy. dankapreomisin), dan membutuhkan setidaknya 2 tahun terapi.Additional drugs include ethionamide (ETH) and para- Obat lain termasuketionamid (ETH) dan ayataminosalicylic (PAS) [ 4 ]. aminosalisilat (PAS) [ 4 ]. Newdrugs with new mechanism Obat baru dengan mekanisme baruof action are urgently needed to shorten the lengthy tindakan sangatdibutuhkan untuk memperpendek panjangtreatment regimen of drug susceptible TB and to improve rejimenpengobatan obat TB rentan dan untuk meningkatkanthe poor cure rates of drug resistant disease [ 5 ]. angkakesembuhan buruk dari penyakit yang resistan terhadap obat [ 5 ].Current drug discovery strategies are largely based on Strategi penemuanobat saat ini sebagian besar didasarkan padaPaul Ehrlich's magic bullet 'one drugone target' con- Paul Ehrlichpeluru ajaib 'satu obat-satu sasaran' concept. kecuali bahwa. This facilitates lead optimization as it allowssimple Ini memfasilitasi optimasi memimpin karena memungkinkan sederhanastructure activity relationships and the use of target- strukturhubungan aktivitas dan penggunaan sasaran daricompound co-structure guided design [ senyawa co-desain struktur dipandu[ 6 6 ]. ]. A few years Beberapa tahunago, after a decade of biochemical high throughput lalu, setelah satudekade throughput yang tinggi biokimiascreening against genetically validated targets, the anti- skriningterhadap target divalidasi genetik, anti tersebutmycobacterial discovery field largely moved back to phe- bidang penemuanmikobakteri sebagian besar pindah kembali ke phenotypic whole cell screens to identify compounds with notypic layarwhole cell untuk mengidentifikasi senyawa denganantimicrobial activity first, then deconvolute the target for aktivitasantimikroba pertama, kemudian deconvolute target untuklead optimization [ 7 ]. optimasi memimpin [ 7 ]. Thisstrategic shift is due to the Pergeseran strategis ini disebabkan olehlarge scale failure of target-based approaches: translating kegagalanskala besar pendekatan berbasis target: menerjemahkanbiochemical enzyme inhibitors into whole cell active inhibitor enzimbiokimia ke seluruh sel aktifantimicrobials turned out to be far more difficult than antimikrobaternyata jauh lebih sulit daripadaanticipated [ 8 ]. diantisipasi [ 8 ]. A key issue is themycobacterial cell Masalah utama adalah sel mikobakterienvelope, representing a formidable permeability barrier amplop,mewakili penghalang permeabilitas tangguh[ [ 9 9 ]. ]. Target based approaches also fail tocapture prodrugs, Pendekatan Sasaran berbasis juga gagal untuk menangkapprodrugs,which are critical components of anti-TB regimens. yang merupakankomponen penting dari rejimen anti-TB. It is Hal inito note that both target-first and compound-first avenues untuk dicatatbahwa jalan kedua target pertama dan senyawa-pertamause the same concept: identify single-target, high affinity menggunakankonsep yang sama: mengidentifikasi target tunggal, afinitas tinggi(nM) binders, which are non-reactive to avoid side (NM) binder, yangnon-reaktif untuk menghindari sisieffects. efek. Other recent approaches do not start with screen-Pendekatan terbaru lainnya tidak mulai dengan penyaringaning of compound libraries, but make use of existing ing perpustakaanmajemuk, tetapi memanfaatkan yang adaantibacterials that either do not work against M. tubercu- antibakteriyang baik tidak bekerja melawan M. tubercu-losis, or are not used for treatment of TB. losis, atau tidak digunakanuntuk pengobatan TB. These include Ini termasukelegant remodelling of antibiotics, such as spectinomycin renovasielegan antibiotik, seperti spectinomycin[ [ 10 ], to make them stay inside the bacillus (prevent 10 ], untuk membuat mereka tinggal di dalam basil (mencegahefflux), and re-purposing of old drugs, such as clofaziminepenghabisan), dan re-pemaknaan dari obat lama, seperti clofazimine[ [ 11 ]. 11 ]. High attrition rates, that is, limitedsuccess in all Angka putus sekolah tinggi, yaitu keberhasilan yangterbatas dalam semuaapproaches, call for a multipronged 'leaving no stone pendekatan,panggilan untuk multipronged 'meninggalkan batuunturned' strategy [ 12 Strategi unturned '[ 12 ]. ]. Canwe find additional hit-finding Dapatkah kita menemukan tambahan hit-temuanavenues at the bottom of the barrel, approaches that we jalan di bagianbawah laras, pendekatan yang kitahave overlooked or not fully utilized so far? telah diabaikan atau belumdimanfaatkan secara maksimal sejauh ini?Some of the key TB drugs, discovered in the middle of Beberapa obat TBkunci, ditemukan di tengah-tengahthe past century by whole cell or animal model screening, abad yang laluoleh sel atau hewan jenis skrining secara keseluruhan,are dirty fragments: they hit multiple targets and their fragmen kotor:mereka memukul beberapa sasaran dan merekamolecular weights are in the range of 100300 g/mol Berat molekul beradadi kisaran 100-300 g / mol( ( Figure 1 ). Gambar 1 ). The fragments are metabolizedinside the Fragmen dimetabolisme di dalamtubercle bacillus and only then, after being 'activated', tuberkulumbacillus dan hanya kemudian, setelah 'diaktifkan',exert their antimicrobial activity [ mengerahkan aktivitas antimikrobamereka [ 13,14 13,14 ]. ]. This type of Jenismechanism of action (MoA), polypharmacology, and phy- Mekanisme kerja(MoA), polypharmacology, dan phy-sicochemical properties, 'extra' small and reactive, is at Sifatsicochemical, 'ekstra' kecil dan reaktif, adalah padaodds with main stream antibacterial drug discovery: bertentangan denganarus utama penemuan obat antibakteri:attractive leads for medicinal chemistry should inhibit lead yangmenarik untuk kimia obat harus menghambatAvailable online at www.sciencedirect.comTersedia online di www.sciencedirect.comScienceDirect ScienceDirectwww.sciencedirect.com www.sciencedirect.comCurrent Opinion in Microbiology 2014, 21:712 Opini Lancar Mikrobiologi2014, 21: 7-12------------------------------------------------------------------------*Page 2* *Page 2*a single target (to facilitate lead optimization), have a target tunggal(untuk memfasilitasi optimasi memimpin), memilikidecent size (to bind a target with high affinity), and should ukuranyang layak (untuk mengikat target dengan afinitas tinggi), dan harusnot be reactive (to avoid side effects) [ 15 tidak reaktif (untukmenghindari efek samping) [ 15 ]. ].Can we learn something from these old TB fragment Bisakah kita belajarsesuatu dari ini fragmen TB tuadrugs? obat? Should we start screening again for these types of Haruskahkita mulai skrining lagi untuk jeniscompounds to identify leads for the discovery of new TB senyawa untukmengidentifikasi lead untuk penemuan TB barudrugs? obat? Here we give an update on some recent develop- Di sini kitamemberikan update pada beberapa pembangunan baru-baru iniments in our understanding of the MoA of some of these KASIH dalampemahaman kita tentang MoA beberapa iniunusual antibiotics, revealing new concepts and targets. antibiotik yangtidak biasa, mengungkapkan konsep dan target baru.Then we zoom into a critical sterilizing (relapse-prevent- Kemudian kamitampilannya menjadi sterilisasi kritis (kambuh-pat dicegahing) TB drug: pyrazinamide (PZA). ing) Obat TB: pirazinamid (PZA). Weargue that the Kami berpendapat bahwaMoA of this metabolized fragment drug remains an MoA ini obat fragmendimetabolisme tetap merupakanenigma, despite some recent progress, and that it is teka-teki, meskipunada beberapa kemajuan baru-baru ini, dan bahwa itu adalahimportant to identify its targets if we are to eliminate penting untukmengidentifikasi target jika kita ingin menghilangkanpersisters and substantially shorten TB therapy. persisters dansubstansial memperpendek terapi TB.We review some key findings from the past few years. Kami meninjaubeberapa temuan kunci dari beberapa tahun terakhir.However, in the case of PZA, we need to go back to the Namun, dalamkasus PZA, kita perlu kembali keearly 1900s, for some apparently forgotten papers. 1900-an, untukbeberapa kertas tampaknya lupa. We Kamipropose that including, or re-introducing, fragment-based mengusulkanbahwa termasuk, atau re-memperkenalkan, berdasarkan fragmen-whole cell screens against replicating and dormant bac- layar seluruhsel terhadap replikasi dan bac- aktifteria in our current approaches to TB drug discovery will teria dalampendekatan kami saat ini untuk penemuan obat TB akandeliver the next generation of TB drugs. memberikan generasi berikutnyadari obat TB.MoA of reactive TB fragment drugs and why it MoA obat fragmen TB reaktifdan mengapais useful to understand them berguna untuk memahami merekaReactivemulti-targetfragmentdrugs,unusuallysmall anti-Reactivemulti-targetfragmentdrugs, unusuallysmall antimycobacterials that are activated inside the bacilli, mycobacterialsyang diaktifkan di dalam basil,represent critical components of TB chemotherapies merupakan komponenpenting dari kemoterapi TB( Figure 1 ( Gambar 1 ). ). INH is the key bactericidal(ie sputum INH adalah bakterisida kunci (yaitu sputumcount-reducing) fragment drug in the first line regimencount-mengurangi) obat fragmen dalam rejimen lini pertama[ 16 ]. [ 16 ]. The compound is oxidized by the bacterialcatalase- Senyawa teroksidasi oleh bakteri catalase-peroxidase KatG [ peroxidase katG [ 17 ], and its reactivemetabolite forms 17 ], dan bentuk-bentuk metabolit reaktifadducts with NAD(P). aduk dengan NAD (P). The enoyl acyl carrier proteinProtein enoyl asil pembawareductase InhA, required for synthesis of outer-membrane reduktase InhA,diperlukan untuk sintesis dari luar-membranmycolicacids,appearstobeits major target[ 18 ].Additionalmycolicacids, appearstobeits sasaran utama [ 18 ] .Additional8 Antimicrobials 8 AntimikrobaFigure 1 Gambar 1*INH* *INH*NH NH2 2NH NH2 2S SNH NH2 2H H2 2N NNH NHN NO OF FH HH HH H3 3C CH H3 3C CH H3 3C CH H3 3C CH H3 3C CH H3 3C CCH CH3 3CH CH3 3CH CH3 3CH CH3 3CH CH3 3CH CH3 3O OO OO OO OO OOH OHO OO OO OO OOH OHHO HOOH OHOH OHOH OHO OO OO OOH OHHO HON NN NNH NHNH NHN NN NN NN NN NN N*ETH* *ETH**PAS* *PAS**PZA* *PZA**MXF* *MXF**RIF* *RIF*Current Opinion in Microbiology Opini Lancar MikrobiologiStructures of TB fragment drugs. Struktur obat fragmen TB. Isoniazid[INH, MW 137], ethionamide [ETH, MW 166], para-aminosalicylic acid [PAS,MW 153] and pyrazinamide Isoniazid [INH, MW 137], etionamid [ETH, MW166], para-aminosalisilat acid [PAS, MW 153] dan pirazinamid[PZA, MW 123] in comparison with a more standard-sized synthetic drug,moxifloxacin [MXF, MW 402], and a huge natural product, rifampicin [RIF,[PZA, MW 123] dibandingkan dengan sintetis obat yang lebih berukuranstandar, moksifloksasin [MXF, MW 402], dan produk alami yang sangatbesar, rifampisin [RIF,MW 823]. MW 823]. MW: molecular weights in g/mol. MW: berat molekuldalam g / mol.Current Opinion in Microbiology 2014, 21:712 Opini Lancar Mikrobiologi2014, 21: 7-12www.sciencedirect.com www.sciencedirect.com------------------------------------------------------------------------*Page 3* *Page 3*targets include dihydrofolate reductase DHFR [ target termasukdihydrofolate reduktase DHFR [ 19 19 ], and ], Danketoacyl acyl carrier protein synthase KasA [ 20 ]. ketoacylsynthase protein pembawa asil Kasa [ 20 ]. At least 16 Setidaknya 16other INH adduct-binding mycobacterial proteins as well INH lainnyaaduk-mengikat protein mikobakteri jugaas several additional known and unknown INH resistant karena beberapatambahan tahan INH dikenal dan tidak dikenalmutations extend the target/MoA list [ 21,22 mutasi memperpanjangtarget / daftar Deptan [ 21,22 ]. ]. ETH, less ETH, kurangwellstudied,isactivatedbythemono-oxygenaseEthA[ wellstudied,isactivatedbythemono-oxygenaseEthA [ 23 23 ] ]and again curiously appears to inhibit InhA as its major dan lagianehnya muncul untuk menghambat InhA sebagai yang utamatarget [ Target [ 24 ]. 24 ]. More recently, the (first?)MoA of PAS was Baru-baru ini, (pertama?) Deptan dari PAS adalahdetermined. ditentukan. The molecule is a structural analogue of theMolekul adalah analog struktural darisubstrate for dihydropteroate synthase (DHPS) in the substrat untuksintase dihidropteroat (DHPS) difolate pathway of mycobacteria. folat jalur mikobakteri. Therefore itwas assumed Oleh karena itu diasumsikanthat PAS acts as a competitive inhibitor of this enzyme. PAS yangbertindak sebagai inhibitor kompetitif enzim ini.However, this is inconsistent with enzymology studies, in Namun, initidak konsisten dengan penelitian enzim, diwhich PAS showed relatively weak inhibitory activity yang PASmenunjukkan aktivitas penghambatan yang relatif lemahagainstDHPS. againstDHPS. Elegantmetabolomics andgenetic analysesElegantmetabolomics analisis andgeneticrevealed that, rather than being (only) an inhibitor of mengungkapkanbahwa, bukannya (hanya) inhibitorDHPS, PAS is recognized as its substrate, resulting in DHPS, PAS diakuisebagai substrat, menghasilkanpoisoning of downstream enzymes of the folate pathway, keracunan enzimhilir dari jalur folat,including dihydrofolate reductase DHFR [ termasuk reduktasedihydrofolate DHFR [ 25 25 ,26 , 26 ]. ]. The Theidentification ofthe MoAof INH, ETH and PAS opens the identifikasitersebut yang MoAof INH, ETH dan PAS membukaway for new drug discovery approaches employing those cara untukpenemuan obat baru pendekatan menggunakan merekanew chemically validated targets. baru divalidasi kimia target. In thecase of INH, for Dalam kasus INH, untukinstance InhA can be directly targeted, circumventing Misalnya InhA bisalangsung ditargetkan, menghindariresistanceduetolossoffunctionmutationsintheactivatingresistanceduetolossoffunctionmutationsintheactivatingenzyme KatG [ 27 ]. enzim katG [ 27 ]. In the case of ETH,efforts are under Dalam kasus ETH, upaya berada di bawahway to inhibit the repressor (EthR) of expression of the cara untukmenghambat represor (EthR) dari ekspresiactivating enzyme EthA [ 28 ]. mengaktifkan enzim Etha [ 28 ].This increases the level of Hal ini meningkatkan tingkatthe activating enzyme and perhaps allows lowering the enzim pengaktifdan mungkin memungkinkan menurunkanETH dose to reduce side effects. Dosis ETH untuk mengurangi efeksamping. The concept of pathway Konsep jalurpoisoning revealed by the PAS findings might be keracunan diungkapkanoleh temuan PAS mungkinapplicable to other biochemical pathways. berlaku untuk jalur biokimialainnya. For the folate Untuk folatpathwayspecifically,targets(forinstanceDHFR)inhibitedpathwayspecifically, target (forinstanceDHFR) menghambatbymetabolizedPASmightbeemployedfortarget-directedbymetabolizedPASmightbeemployedfortarget-diarahkandesignapproaches[ designapproaches [ 25 25 ,26 , 26 ].Thisshowsthatunderstand- ] .Thisshowsthatunderstand-ing the MoA of fragments does deliver new concepts and ing MoA fragmentidak memberikan konsep-konsep baru dantargets which can be exploited for drug discovery. target yang dapatdimanfaatkan untuk penemuan obat.The pyrazinamide enigma and why we need to The pirazinamid enigma danmengapa kita perluelucidate it menjelaskan ituShortening treatment time while improving relapse rates Memperpendekwaktu pengobatan sementara meningkatkan tingkat kambuhanis the major objective in TB drug development [ adalah tujuan utamadalam pengembangan obat TB [ 5 5 ]. ]. To Untukreach that goal, understanding why current therapies mencapai tujuanitu, pemahaman terapi mengapa saat inimust be administered for 624 months and more, while harus diberikanselama 6-24 bulan dan lebih, sementaramost antimycobacterials with the exception of kebanyakanantimycobacterials - dengan pengecualianPZA inhibit and/or kill bacteria in vitro rather rapidly, PZA -menghambat dan / atau membunuh bakteri in vitro agak cepat,is a key starting point. adalah titik awal kunci. Many workinghypotheses, falling Banyak hipotesis kerja, jatuhmostly into two categories, have been developed to sebagian besar kedalam dua kategori, telah dikembangkan untukexplain treatment duration. menjelaskan durasi pengobatan. The drugs mayfail to reach Obat-obatan mungkin gagal untuk mencapaitheir cellular target, the bacilli, which are hiding in deep Targetselular mereka, basil, yang bersembunyi di dalamtissue inside various, some poorly vascularized, lesion jaringan didalam berbagai, beberapa buruk vascularized, lesitypes [ jenis [ 29 ]. 29 ]. Alternatively, subpopulationsof in vivo bacilli Atau, sub-populasi dari in vivo basilmight be physiologically different from in vitro growing mungkinfisiologis berbeda dari in vitro tumbuhbacteria, against which we test our compounds. bakteri, yang kamimenguji senyawa kami. They Merekacould exist in phenotypically drug resistant, for instance bisa adadalam obat fenotip tahan, misalnyanon-replicating, forms [ 9,30,31 non-replikasi, bentuk [ 9,30,31 ]. ]. There is limited evi- Ada bukti-terbatasdence to support the pharmacokinetic (PK) and the dence untuk mendukungfarmakokinetik (PK) danbacteriological models of persistence, but so far we do modelbakteriologis ketekunan, tapi sejauh ini kami lakukannot have clear, clinical answers. tidak memiliki jelas, jawaban klinis.What we know is that inclusion of PZA in the current first Apa yang kitatahu adalah bahwa dimasukkannya PZA di saat pertamaline regimen many decades ago resulted in shortening of rejimen linibeberapa dekade yang lalu mengakibatkan pemendekantreatment from nine to six months. pengobatan 9-6 bulan. PZA is alsoconsidered PZA juga dianggapa key sterilizing and relapse-preventing drug [ 32 a sterilisasikunci dan kambuh-mencegah narkoba [ 32 ], indi- ],-Individucating that it must target persisting mycobacterial popu- cating yangharus menargetkan bertahan popu- mikobakterilations that are not killed by other drugs. lations yang tidak dibunuholeh obat lain. Thus, learning Dengan demikian, belajarfrom PZA's mechanism of action, unique treatment short- dari mekanismePZA tentang tindakan, pendek pengobatan yang unikening properties, and lesion penetration characteristics, propertiEning, dan karakteristik penetrasi lesi,seems to be the logical path to rationally discover drugs tampaknyamenjadi jalur logis untuk menemukan obat secara rasionalthat shorten TB therapy. yang mempersingkat terapi TB. It may also helpcharacterize key Hal ini juga dapat membantu kunci cirifunctions of persisting mycobacteria and refine the envi- fungsibertahan mycobacteria dan memperbaiki vironment yangronment or assay conditions adopted in screening cam- kondisi ronmentatau uji diadopsi dalam penyaringan cam-paigns. paigns. While we know that M. tuberculosis undergoes Sementarakita tahu bahwa M. tuberculosis mengalamimetabolic remodelling and physiological adaptation in renovasimetabolisme dan adaptasi fisiologis dalamresponse to microenvironments present in TB lesions Menanggapimicroenvironments hadir dalam lesi TB[ [ 9 9 ], adequate recapitulation of theseconditions for ], Rekapitulasi yang memadai atas kondisi tersebuthigh-throughput screening against persisters is mostly screeningtinggi-throughput terhadap persisters sebagian besarmatter for debate. masalah untuk diperdebatkan. Defining conditionsunder which Kondisi Mendefinisikan di manaPZA kills in vitro as it does in vivo would provide much PZA membunuh invitro seperti halnya in vivo akan memberikan banyakneeded fresh approaches to TB drug discovery. diperlukan pendekatansegar untuk penemuan obat TB. Ironically, Ironisnya,PZA is probably the least understood fragment drug. PZA mungkin yangpaling sedikit dipahami obat fragmen.What is known about the MoA of PZA? Apa yang diketahui tentang MoA dariPZA? Clinical and in Klinis dan divitro resistance is largely caused by mutations in the resistensi vitrosebagian besar disebabkan oleh mutasi padanicotinamidase gene pncA [ gen nicotinamidase pncA [ 33 ]. 33 ]. The amidase generates Amidase menghasilkanwhat appears to be the active component pyrazinoic acid apa yangtampaknya menjadi asam pyrazinoic komponen aktif(POA). (POA). POA/PZA has multiple MoAs. POA / PZA memiliki beberapaMOAs. Under acidic cul- Dalam kultur asamture conditions the weak acid POA affects the bacterial kondisimendatang POA asam lemah mempengaruhi bakterimembrane potential [ membran potensial [ 34 ]. 34 ]. POA(and PZA) inhibits fatty POA (dan PZA) menghambat lemakacid synthase (FAS) I [ 3537 ]. synthase asam (FAS) I [ 35-37 ]. Via an affinity purification Melalui pemurnian afinitasapproach, several POA binding proteins were identified, pendekatan,beberapa protein POA mengikat diidentifikasi,and it was shown that POA interferes with trans-trans- dan itumenunjukkan bahwa POA mengganggu trans-translation, a bacterial rescue mechanism for ribosomes stuck lation,mekanisme penyelamatan bakteri untuk ribosom terjebakduring translation, by binding to the ribosomal S1 protein selamaterjemahan, dengan mengikat protein ribosom S1[ [ 38 38 ]. ]. Recently, mutations in the aspartate decarboxylase Baru-baru ini,mutasi pada dekarboksilase aspartatPanD, required for synthesis of pantothenate, were found Pand,diperlukan untuk sintesis pantothenate, ditemukanto correlate with PZA resistance [ 39 berkorelasi dengan resistensiPZA [ 39 ]. ]. The identity of Identitasother PncA wild type but PZA resistant mutants remains lainnya PncAJenis liar tapi PZA mutan tahan sisa-sisato be determined, suggesting additional targets and MoAs ditentukan,menunjukkan target tambahan dan MOAsof PZA [ dari PZA [ 39 ]. 39 ]. It is to note thatattempts to isolate POA Hal ini untuk dicatat bahwa mencoba untukmengisolasi POAresistant tubercle bacilli directly in vitro were unsuccess- tahan basiltuberkulosis langsung in vitro yang unsuccess-ful [ ful [ 35,40 35,40 ]. ]. Assuming that POA isindeed the critical Dengan asumsi bahwa POA memang kritisantimicrobial metabolite, this suggests that work remains metabolitantimikroba, ini menunjukkan bahwa pekerjaan yangto be done to unravel the complete MoA of PZA. harus dilakukan untukmengungkap MoA lengkap PZA.A major hurdle in further elucidating PZA's MoA is the Sebuah rintanganutama dalam lebih mengelusidasi PZA ini Deptan adalahuncertainty around the predictive value of in vitro cultureketidakpastian nilai prediktif kultur in vitroconditions under which we measure PZA's antimicrobial kondisi di manakita mengukur antimikroba PZA iniactivity. aktivitas. This makes target deconvolution difficult at best.Hal ini membuat Target dekonvolusi sulit di terbaik.PZA was discovered due to its activity in mice [ 41,42 PZAditemukan karena aktivitasnya pada tikus [ 41,42 ] ]rather than in a test tube under standard conditions. bukan dalam tabungreaksi dalam kondisi standar. Soon Segerathe compound was found to be clinically efficacious [ 43 Senyawaitu ditemukan secara klinis berkhasiat [ 43 ], ],which led to renewed attempts to identify in vitro yang menyebabkanupaya baru untuk mengidentifikasi in vitroconditions under which it was active. kondisi di mana itu aktif. In the1950s, Pada tahun 1950,McDermott and Tompsett showed that PZA exhibits McDermott dan Tompsettmenunjukkan bahwa PZA pameranReactive fragments for TB drug discovery Gopal and Dick 9 Fragmenreaktif untuk penemuan obat TB Gopal dan Dick 9www.sciencedirect.com www.sciencedirect.comCurrent Opinion in Microbiology 2014, 21:712 Opini Lancar Mikrobiologi2014, 21: 7-12------------------------------------------------------------------------*Page 4* *Page 4*better growth inhibitory activity in vitro under mildly acid aktivitaspenghambatan pertumbuhan yang lebih baik in vitro bawah agak asamconditions (while standard media have neutral pH) [ kondisi (sementaramedia yang standar memiliki pH netral) [ 44 ]. 44 ].This led to the belief that TB lesions are acidic, at least in Hal inimenyebabkan keyakinan bahwa lesi TB bersifat asam, setidaknya dalamlocalized areas, providing a rational explanation for the daerah lokal,memberikan penjelasan yang rasional untuksurprising activity of PZA in vivo [ Kegiatan mengejutkan PZA in vivo [ 32,45 32,45 ]. ]. However, some Namun, beberapaearly apparently forgotten experimental data on the awal - tampaknyalupa - data eksperimen padapH of TB lesions from 1934, suggest that lesions are pH lesi TB daritahun 1934, menunjukkan bahwa lesiactually not acidic [ 46,47 ]. sebenarnya tidak asam [ 46,47 ]. In fact, the growth inhibition Bahkan, penghambatan pertumbuhandata of McDermott and Tompsett do not show that PZA data McDermott danTompsett tidak menunjukkan PZA yangis active only under acid conditions. hanya aktif dalam kondisi asam.They reveal a Mereka mengungkapkanU-shaped pH activity curve, where potency decreases U-berbentuk kurvaaktivitas pH, di mana potensi menurunfrom acidic to neutral pH and then increases again under dari asam ke pHnetral dan kemudian meningkat lagi di bawahmild alkaline conditions [ kondisi alkali ringan [ 44 ]. 44 ]. Is our in vitro culture assay Apakah in vitro assay budaya kitaadequate, does it reflect in vivo conditions? memadai, apakah itumencerminkan kondisi vivo? Have we been Apakah kita pernahchasing a PZA-pH myth for the past half century? mengejar mitos PZA-pHuntuk setengah abad terakhir?In our opinion, two approaches that have been neglected Menurut pendapatkami, dua pendekatan yang telah diabaikanthus far would help unravel the PZA enigma: first, careful sejauh iniakan membantu mengungkap teka-teki PZA: pertama, hati-hatiand standardized measurement of the actual pH of necro- dan pengukuranstandar dari pH aktual nekrotikanstic lesions, across animal models and man, and second, lesi tic, seluruhmodel hewan dan manusia, dan kedua,isolation of POA resistant mutants to understand the dirty isolasi mutantahan POA untuk memahami kotordrug characteristics of PZA and how they relate to its karakteristikobat PZA dan bagaimana mereka berhubungan dengan perusahaansterilizing properties. mensterilkan properti.Why fragment-based whole cell screens might Mengapa layar seluruh selberbasis fragmen mungkinbe useful bergunaFragments cameto famein modern drugdiscovery dueto a Fragmen cametodrugdiscovery famein modern yang dueto sebuahnovel, fragment-based screening approach against molecu- Novel,pendekatan skrining berbasis fragmen terhadap molecu-lar targets [ target lar [ 48 48 ]. ]. This methodfor lead finding makes use of Metode ini untuk menemukan memimpinmemanfaatkansmall libraries of promiscuous binders: very small mol- perpustakaankecil pengikat promiscuous: mol sangat kecilecules ('fragments'), with weak binding affinities. ecules ('fragmen'),dengan afinitas mengikat lemah. Bio- Biophysical and structural biology methods are employed to metode biologifisik dan struktur yang digunakan untukidentify such high mM/mM binders to a particular target.mengidentifikasi tinggi seperti binder mM / mM ke target tertentu.The weak binders are then extended or combined to Pengikat lemahkemudian diperpanjang atau dikombinasikan untukdevelop tight, target-specific, high affinity (nM) com- mengembangkanketat,-target khusus, afinitas tinggi (nM) compounds. pound. Fragments usually represent attractive starting Fragmenbiasanya merupakan menarik mulaipoints for medicinal chemistry as they have preferred poin untuk kimiaobat karena mereka lebih sukaphysicochemical and PK properties: they are relatively fisikokimia dansifat PK: mereka relatifsmall (