Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.
İlaç metabolizması ve inflamasyon kritik bir düzenleyicisi olarak, pregnan X Reseptör (PX), metabolizma ve enflamasyon (örneğin, karaciğer yağlanması) 1,2 bağlama hastalığın patofizyolojisinde önemli bir rol oynar. PX için agonist ligandların belirlenmesinde çok ilerleme olmuştur Bununla birlikte, ilaç-benzeri antagonistler ve PX 3,4,5 üzerindeki bağlanma siteleri sınırlı açıklamaları vardır. Kritik bir bariyer verimli PX 1998 klonlandı ve karakterize edildi rağmen antagonistleri ile yapı çalışmaları için de tam uzunluktaki proteini saflaştırmak için yetersizlik olmuştur. Bizim laboratuar PX 6 artıkları bağlayıcı, bir antagonist, ketokonazol en tanımlamak için temel alan yeni yüksek kapasiteli maya iki-hibrid tahlili geliştirilmiştir. Bizim yöntem ketokonazol ile etkileşim beklenen pxr ve AF-2 yüzeyinde tek mutasyonların etkisini kurtarırdı mutasyon kütüphanelerini oluşturma içerir. Kurtarma veya "kazanç fonksiyon-" sekond mutasyonlar PX üzerinde ketokonazol genetik etkileşim ve kalıntı yüzey (ler) ile ilgili sonuçlar, uygun olduğu şekilde yapılabilir. Böylece, kendi koaktivatörle, SRC-1 ile etkileşim PX mutantlarının veren yüksek işleme kapasiteli, iki maya hibrid ekran geliştirdi. Maya antifungal ilaç, ketokonazol çalışma karşılamak için modifiye edildiği, bu yaklaşım kullanarak, ketokonazol bağlamak için mümkün klonlarında zenginleştirilmiş PX belirli mutasyon göstermek olabilir. Ters mantıkla, biz orijinal kalıntıları ketokonazol ile doğrudan etkileşim kalıntıları olduğu sonucuna. Bu deney, bir yeni, nükleer reseptör yüzeyler üzerinde antagonist bağlanma bölgeleri için ekrana uysal genetik deneyi temsil etmektedir. Bu deney, maya hem de standart yapısal biyoloji veya proteomik göre yöntemler kullanılarak incelendi edilemez hücresel protein (ler) bağımsız olarak bir sitotoksik potansiyel herhangi bir ilaç için uygulanabilir. Potansiyel tuzaklar verilerin yorumlanmasını (tamamlayıcı yöntemler yararlı), dinleri dahilTek Y2H yöntemi, maya taşıma veya maya iki hibrit deneyleri performans uzmanlık ve tahlil optimizasyonu ance.
The yeast two-hybrid (Y2H) assay is widely used to discover protein-protein interactions and more recently for discovery of novel small molecules that disrupt protein-protein interaction complexes 7, 8, 9, 10, 11. However, the conventional approaches of this assay, used for drug discovery or "hits", do not allow for detection of allosteric interaction residues of chemicals compounds within protein-protein surfaces, that when altered still interact and allow for interrogation of the altered residues11. Indeed, such a method(s), if feasible to develop, would enable a tractable yeast system for high throughput assessment of allosteric interaction residues critical for protein-protein interaction disruption. In the context of drug discovery, the most direct way to establish interaction of compounds with proteins would involve structural determination (e.g. crystalization of protein-inhibitor complex). These methods are cumbersome, use elaborate resources and it is not technically feasible to perform structural studies on every protein.
Tractable genetic drug screening systems have been established in bacteria1, 2 and other model systems like mammalian two-hybrid. However, these systems need optimization and alternative systems like Y2H are still the most tested in drug discovery. There are limitations that include poor sensitivity and reliability of interactions using singular methods13 , however, a single Y2H assay can be modified to answer specific questions regarding interaction residues. In the field of nuclear receptor research, Y2H has been used to define interacting proteins14, however, these protein interactions have rarely been used to define the nature in which ligands/antagonists interact with nuclear receptor-protein complexes. Thus, our laboratory focused efforts on defining a method, especially for receptor proteins that are not readily amenable to proteomic based methods, that would unearth novel ligand/antagonist interacting residues using a reverse Y2H based discovery platform.
Based on our previous finding that ketoconazole disrupts PXR and its activator SRC-1, we developed a novel reverse Y2H system that enable us to define and interrogate ketoconazole interacting residues on PXR6. Our method is based on the properties of the yeast GAL4 protein that consists of separable domains responsible for DNA-binding and transcriptional activation. The PXR LBD protein is expressed as a fusion to the LexA DNA-binding domain (DNA-BD), while the full length co-activators SRC-1 (steroid receptor coactivator 1) proteins are expressed as fusions to the GAL4 activation domain (AD). Interaction between PXR and SRC-1 fusion proteins leads to the transcriptional activation of GAL4-binding sites containing reporter gene β-LacZ that is integrated into the yeast genome. Ketoconazole, a PXR antagonist, disrupts PXR and SRC-1 interaction 15, 16, 17 and we can detect the interaction of PXR and SRC-1 in the presence or absence of ketoconazole after staining colonies on filters for X-gal activity. The principle of Y2H is illustrated in Figure 1 and the experimental procedure is summarized in Figure 2.
In our modified Y2H assay, we have identified important residues for ketoconazole interactions on PXR6. Since SRC-1 is a coactivator (and was cloned into the pGADNot vector), we also tested whether SRC-1 could activate lacZ expression when cloned into the pSH vector system and whether this would change the activation profile and/or affect the leakiness of the yeast two-hybrid assay. Using our redesigned plasmids we performed two-hybrid assays in erg3Δ/erg11Δ yeast. As before, we sho…
The authors have nothing to disclose.
This work was supported by National Institutes of Health (NIH) Grants CA127231 and The Damon Runyon Foundation Clinical Investigator Award (CI 1502) (to S.M). We would like to thank Professor Zdenek Dvorak from Palacky University Olomouc, Czech Republic for his helpful insights into discussing portability of this technique to their institution and standardization of protocol.
Name | Company | Catalog Number | Comments |
Yeast Strain CTY10-5d erg3Δ/erg11Δ | Our lab | CTY10-5d yeast was double knocked out ERG3 and ERG11 (erg3Δ/erg11Δ) genes6 . | |
YPD Growth Medium | BD Biosciences | 630409 | |
Difco Yeast Nitrogen Base (YNB) w/o Amino Acids and Ammonium Sulfate | BD Biosciences | 233520 | |
Bacto Agar | BD Biosciences | 214010 | |
CSM-His/-Leu Complete Supplement Mixture | MP Biomedicals | 4250-412 | |
ONPG (o-Nitrophenyl Β-D- Galactopyranoside). | Sigma-Aldrich | N1127 | |
2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
Luria Broth (LB) | Sigma-Aldrich | L3022 | |
X-Gal | Fisher | BP-1615 | |
Sonicated Salmon Sperm DNA boiled (10 mg/ml) | Life Technology | 156-017 | |
Ampicillin | Acros Organics | 61177 | |
Ketoconazole | Sigma-Aldrich | K1003 | |
N,N-Dimethylformamide | Acros Organics | 326871000 | |
Lithium Acetate | Sigma-Aldrich | L4158 | |
50% PEG-3350 solution, filter-sterilized | Sigma-Aldrich | P-3640 | |
Nitrocellulose Membrane | Whatman | 10402091 | |
10 cm Petri Dish | Fisher | 875712 | |
5'-ACCGGATCCCGATGAAGA AGGAGATGATCATGTCC-3' | our lab | PXR LBD forward primer for pSH2-1 | |
5'-AGAGTCGACTCAGCTA CCTGTGATGCC -3' | our lab | PXR LBD reverse primer for pSH2-1 | |
5'-TATAGC GGCCGCATGAGTG GCCTCGGGGACAGTTCATCC -3' | our lab | SRC-1 forward primer for pGADNOT | |
5'-GCGGTCGACTTATTCAGTCA GTAGCTG -3' | our lab | SRC-1 reverse primer for pGADNOT | |
Platinum PCR Supermix | Invitrogen | 11306-016 | |
BamHI | our lab | R0136 | |
SalI | our lab | R0138 | |
NotI | our lab | R0189 |