AX20017
目录号 : GC32196
AX 20017 is a small-molecule protein kinase G (PknG) inhibitor with an IC50 of 0.39 μM.
Cas No.:329221-38-7
Sample solution is provided at 25 µL, 10mM.
;)
,-1-7$$$37316672.jpg');)
;)
;)
$$$36806353.jpg');)
;33(7)%EF%BC%9A546-561$$$37156877.jpg');)
;)
;)
;)
%201124-1138.$$$35908550.jpg');)
%20766-780.$$$37100057.jpg');)
%20418-432.$$$36893736.jpg');)
%EF%BC%9A-240-255.$$$35108512.jpg');)
533-545$$$36543525.jpg');)
%201-13.$$$36600053.jpg');)
;)
Quality Control & SDS
- View current batch:
- Purity: >99.50%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Kinase experiment: | In vitro phosphorylation by PknG (0.5 μg) is in 25 mM Tris (pH 7.5), 2 mM MnCl2, and 0.5 μCi [γ-32P]ATP in the absence or presence of the reagents. To monitor kinase activity of PknGΔN, the protein is combined with equal amounts of the kinase-dead mutant of full-length PknG, PknG-K181M. To analyze kinase activity of PknG-I87S/A92S and PknG-C/S, the PknG-N-terminal fragment of PknG (2 μg) is included. Phosphorylated proteins are separated on 12.5% SDS/PAGE and analyzed by autoradiography or quantitated by PhosphorImage analysis. IC50 values are determined by using a radiometric ATP consumptive assay. Twelve concentrations of AX20017 in the range from 5 × 10-5M to 1.5 × 10-10 M are tested in each kinase assay[3]. |
Cell experiment: | Phagocytosis is analyzed after incubation of J774 cells for 30 min in the presence of the indicated concentration of AX20017 (0, 10, 20 μM), followed by incubating the cells for 2 h with latex beads at a ratio of 10:1 beads/cells in the continued presence of the inhibitor, followed by fixation in 3% paraformaldehyde as described. Cells are observed with a Axiophot using a ×63 objective. Proliferation of J774 cells is analyzed by incorporation of tritiated thymidine (0.1 μCi) for 12 h as described of cells that had been incubated for 48 h in the absence or presence of the AX20017(0, 10, 20 μM)[3]. |
References: [1]. Walburger A, et al. Protein kinase G from pathogenic mycobacteria promotes survival within macrophages. Science. 2004 Jun 18;304(5678):1800-4. | |
AX 20017 is a small-molecule protein kinase G (PknG) inhibitor with an IC50 of 0.39 μM.
[1] Walburger A, et al. Science. 2004 Jun 18;304(5678):1800-4.
| Cas No. | 329221-38-7 | SDF | |
| Canonical SMILES | O=C(N)C1=C(NC(C2CC2)=O)SC3=C1CCCC3 | ||
| 分子式 | C13H16N2O2S | 分子量 | 264.34 |
| 溶解度 | DMSO : ≥ 32 mg/mL (121.06 mM) | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
||
| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 3.783 mL | 18.915 mL | 37.8301 mL |
| 5 mM | 0.7566 mL | 3.783 mL | 7.566 mL |
| 10 mM | 0.3783 mL | 1.8915 mL | 3.783 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Deletion of pknG Abates Reactivation of Latent Mycobacterium tuberculosis in Mice
Antimicrob Agents Chemother 2021 Mar 18;65(4):e02095-20.PMID:33468473DOI:10.1128/AAC.02095-20.
Eradication of tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been a challenge due to its uncanny ability to survive in a dormant state inside host granulomas for decades. Mtb rewires its metabolic and redox regulatory networks to survive in the hostile hypoxic and nutrient-limiting environment, facilitating the formation of drug-tolerant persisters. Previously, we showed that protein kinase G (PknG), a virulence factor required for lysosomal escape, aids in metabolic adaptation, thereby promoting the survival of nonreplicating mycobacteria. Here, we sought to investigate the therapeutic potential of PknG against latent mycobacterium. We show that inhibition of PknG by AX20017 reduces mycobacterial survival in in vitro latency models such as hypoxia, persisters, and nutrient starvation. Targeting PknG enhances the bactericidal activity of the frontline anti-TB drugs in peritoneal macrophages. Deletion of pknG resulted in 5- to 15-fold-reduced survival of Mtb in chronically infected mice treated with anti-TB drugs. Importantly, in the Cornell mouse model of latent TB, the deletion of pknG drastically attenuated Mtb's ability to resuscitate after antibiotic treatment compared with wild-type and complemented strains. This is the first study to investigate the sterilizing activity of pknG deletion and inhibition for adjunct therapy against latent TB in a preclinical model. Collectively, these results suggest that PknG may be a promising drug target for adjunct therapy to shorten the treatment duration and reduce disease relapse.
Flavanones: A potential natural inhibitor of the ATP binding site of PknG of Mycobacterium tuberculosis
J Biomol Struct Dyn 2022;40(22):11885-11899.PMID:34409917DOI:10.1080/07391102.2021.1965913.
Over the years, Mycobacterium tuberculosis has been one of the major causes of death worldwide. As several clinical isolates of the bacteria have developed drug resistance against the target sites of the current therapeutic agents, the development of a novel drug is the pressing priority. According to recent studies on Mycobacterium tuberculosis, ATP binding sites of Mycobacterium tuberculosis serine/threonine protein kinases (MTPKs) have been identified as the new promising drug target. Among the several other protein kinases (PKs), Protein kinase G (PknG) was selected for the study because of its crucial role in modulating bacterium's metabolism to survive in host macrophages. In this work, we have focused on the H37Rv strain of Mycobacterium tuberculosis. A list of 477 flavanones obtained from the PubChem database was docked one by one against the crystallized and refined structure of PknG by in-silico techniques. Initially, potential inhibitors were narrowed down by preliminary docking. Flavanones were then selected using binding energies ranging from -7.9 kcal.mol-1 to -10.8 kcal.mol-1. This was followed by drug-likeness prediction, redocking analysis, and molecular dynamics simulations. Here, we have used experimentally confirmed drug AX20017 as a reference to determine candidate compounds that can act as potential inhibitors for PknG. PubChem165506, PubChem242065, PubChem688859, PubChem101367767, PubChem3534982, and PubChem42607933 were identified as possible target site inhibitors for PknG with a desirable negative binding energy of -8.1, -8.3, -8.4, -8.8, -8.6 and -7.9 kcal.mol-1 respectively. Communicated by Ramaswamy H. Sarma.
Structural basis for the specific inhibition of protein kinase G, a virulence factor of Mycobacterium tuberculosis
Proc Natl Acad Sci U S A 2007 Jul 17;104(29):12151-6.PMID:17616581DOI:10.1073/pnas.0702842104.
The pathogenicity of mycobacteria such as Mycobacterium tuberculosis is closely associated with their capacity to survive within host macrophages. A crucial virulence factor for intracellular mycobacterial survival is protein kinase G (PknG), a eukaryotic-like serine/threonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG with the highly selective low-molecular-weight inhibitor AX20017 results in mycobacterial transfer to lysosomes and killing of the mycobacteria. Here, we report the 2.4 A x-ray crystal structure of PknG in complex with AX20017. The unique multidomain topology of PknG reveals a central kinase domain that is flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Directed mutagenesis suggests that the rubredoxin domain functions as a regulator of PknG kinase activity. The structure of PknG-AX20017 further reveals that the inhibitor is buried deep within the adenosine-binding site, targeting an active conformation of the kinase domain. Remarkably, although the topology of the kinase domain is reminiscent of eukaryotic kinases, the AX20017-binding pocket is shaped by a unique set of amino acid side chains that are not found in any human kinase. Directed mutagenesis of the unique set of residues resulted in a drastic loss of the compound's inhibitory potency. Our results explain the specific mode of action of AX20017 and demonstrate that virulence factors highly homologous to host molecules can be successfully targeted to block the proliferation of M. tuberculosis.
Insights from the molecular docking of withanolide derivatives to the target protein PknG from Mycobacterium tuberculosis
Bioinformation 2011;7(1):1-4.PMID:21904430DOI:10.6026/97320630007001.
A crucial virulence factor for intracellular Mycobacterium tuberculosis survival is Protein kinase G (PknG), a eukaryotic-like serinethreonine protein kinase expressed by pathogenic mycobacteria that blocks the intracellular degradation of mycobacteria in lysosomes. Inhibition of PknG results in mycobacterial transfer to lysosomes. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show various pharmacological activities. We describe the docking of 26 withanferin and 14 withanolides from Withania somnifera into the three dimensional structure of PknG of M. tuberculosis using GLIDE. The inhibitor binding positions and affinity were evaluated using scoring functions- Glidescore. The withanolide E, F and D and Withaferin - diacetate 2 phenoxy ethyl carbonate were identified as potential inhibitors of PknG. The available drug molecules and the ligand AX20017 showed hydrogen bond interaction with the aminoacid residues Glu233 and Val235. In addition to Val235 the other amino acids, Gly237, Gln238 and Ser239 are important for withanolide inhibitor recognition via hydrogen bonding mechanisms.
Identifying RO9021 as a Potential Inhibitor of PknG from Mycobacterium tuberculosis: Combinative Computational and In Vitro Studies
ACS Omega 2022 May 31;7(23):20204-20218.PMID:35721990DOI:10.1021/acsomega.2c02093.
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). Despite being considered curable and preventable, the increase of antibiotic resistance is becoming a serious public health problem. Mtb is a pathogen capable of surviving in macrophages, causing long-term latent infection where the mycobacterial serine/threonine protein kinase G (PknG) plays a protective role. Therefore, PknG is an important inhibitory target to prevent Mtb from entering the latency stage. In this study, we use a pharmacophore-based virtual screening and biochemical assays to identify the compound RO9021 (CHEMBL3237561) as a PknG inhibitor. In detail, 1.5 million molecules were screened using a scalable cloud-based setup, identifying 689 candidates, which were further subjected to additional screening employing molecular docking. Molecular docking spotted 62 compounds with estimated binding affinities of -7.54 kcal/mol (s.d. = 0.77 kcal/mol). Finally, 14 compounds were selected for in vitro experiments considering previously reported biological activities and commercial availability. In vitro assays of PknG activity showed that RO9021 inhibits the kinase activity similarly to AX20017, a known inhibitor. The inhibitory effect was found to be dose dependent with a relative IC50 value of 4.4 卤 1.1 渭M. Molecular dynamics simulations predicted that the PknG-RO9021 complex is stable along the tested timescale. Altogether, our study indicates that RO9021 is a noteworthy drug candidate for further developing new anti-TB drugs that hold excellent reported pharmacokinetic parameters.