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Molecular Pharmacology
Journal Prestige (SJR): 2.076  Citation Impact (citeScore): 4 Number of Followers: 2  Hybrid journal (It can contain Open Access articles)ISSN (Online) 1521-0111 Published by ASPET  [4 journals]
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- CRISPR/Cas9-Mediated Induction of Relapse-Specific NT5C2 and PRPS1
Mutations Confers Thiopurine Resistance as a Relapsed Lymphoid Leukemia
Model [Articles]-
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Authors: Nguyen, T. T. T; Tanaka, Y, Sanada, M, Hosaka, M, Tamai, M, Kagami, K, Komatsu, C, Somazu, S, Harama, D, Kasai, S, Watanabe, A, Akahane, K, Goi, K, Inukai, T.
Pages: 199 - 210
Abstract: 6-Mercaptopurine (6-MP) is a key component in maintenance therapy for childhood acute lymphoblastic leukemia (ALL). Recent next-generation sequencing analysis of childhood ALL clarified the emergence of the relapse-specific mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism. In this scenario, minor clones of leukemia cells could acquire the 6-MP–resistant phenotype as a result of the NT5C2 or PRPS1 mutation during chemotherapy (including 6-MP treatment) and confer disease relapse after selective expansion. Thus, to establish new therapeutic modalities overcoming 6-MP resistance in relapsed ALL, human leukemia models with NT5C2 and PRPS1 mutations in the intrinsic genes are urgently required. Here, mimicking the initiation process of the above clinical course, we sought to induce two relapse-specific hotspot mutations (R39Q mutation of the NT5C2 gene and S103N mutation of the PRPS1 gene) into a human lymphoid leukemia cell line by homologous recombination (HR) using the CRISPR/Cas9 system. After 6-MP selection of the cells transfected with Cas9 combined with single-guide RNA and donor DNA templates specific for either of those two mutations, we obtained the sublines with the intended NT5C2-R39Q and PRPS1-S103N mutation as a result of HR. Moreover, diverse in-frame small insertion/deletions were also confirmed in the 6-MP–resistant sublines at the target sites of the NT5C2 and PRPS1 genes as a result of nonhomologous end joining. These sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations in the 6-MP sensitivity and development of therapy overcoming the thiopurine resistance of leukemia cells.SIGNIFICANCE STATEMENTMimicking the initiation process of relapse-specific mutations of the NT5C2 and PRPS1 genes in childhood acute lymphoblastic leukemia treated with 6-mercaptopurine (6-MP), this study sought to introduce NT5C2-R39Q and PRPS1-S103N mutations into a human lymphoid leukemia cell line by homologous recombination using the CRISPR/Cas9 system. In the resultant 6-MP–resistant sublines, the intended mutations and diverse in-frame small insertions/deletions were confirmed, indicating that the obtained sublines are useful for molecular pharmacological evaluation of the NT5C2 and PRPS1 gene mutations.
PubDate: 2023-03-16T14:37:28-07:00
DOI: 10.1124/molpharm.122.000546
Issue No: Vol. 103, No. 4 (2023)
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- The Small Molecule Antagonist KCI807 Disrupts Association of the
Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the
Adjacent DNA Binding Domain [Articles]-
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Authors: Soave, C; Ducker, C, Islam, N, Kim, S, Yurgelevic, S, Nicely, N. I, Pardy, L, Huang, Y, Shaw, P. E, Auner, G, Dickson, A, Ratnam, M.
Pages: 211 - 220
Abstract: The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD.SIGNIFICANCE STATEMENTThe small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.
PubDate: 2023-03-16T14:37:28-07:00
DOI: 10.1124/molpharm.122.000589
Issue No: Vol. 103, No. 4 (2023)
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- Use-Dependent Relief of Inhibition of Nav1.8 Channels by A-887826
[Articles]-
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Authors: Jo, S; Zhang, H.-X. B, Bean, B. P.
Pages: 221 - 229
Abstract: Sodium channel inhibitors used as local anesthetics, antiarrhythmics, or antiepileptics typically have the property of use-dependent inhibition, whereby inhibition is enhanced by repetitive channel activation. For targeting pain, Nav1.8 channels are an attractive target because they are prominent in primary pain-sensing neurons, with little or no expression in most other kinds of neurons, and a number of Nav1.8-targeted compounds have been developed. We examined the characteristics of Nav1.8 inhibition by one of the most potent Nav1.8 inhibitors so far described, A-887826, and found that when studied with physiologic resting potentials and physiologic temperatures, inhibition had strong "reverse use dependence", whereby inhibition was relieved by repetitive short depolarizations. This effect was much stronger with A-887826 than with A-803467, another Nav1.8 inhibitor. The use-dependent relief from inhibition was seen in both human Nav1.8 channels studied in a cell line and in native Nav1.8 channels in mouse dorsal root ganglion (DRG) neurons. In native Nav1.8 channels, substantial relief of inhibition occurred during repetitive stimulation by action potential waveforms at 5 Hz, suggesting that the phenomenon is likely important under physiologic conditions.SIGNIFICANCE STATEMENTNav1.8 sodium channels are expressed in primary pain-sensing neurons and are a prime current target for new drugs for pain. This work shows that one of the most potent Nav1.8 inhibitors, A-887826, has the unusual property that inhibition is relieved by repeated short depolarizations. This "reverse use dependence" may reduce inhibition during physiological firing and should be selected against in drug development.
PubDate: 2023-03-16T14:37:28-07:00
DOI: 10.1124/molpharm.122.000593
Issue No: Vol. 103, No. 4 (2023)
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- Genetic Knockout of Fatty Acid Amide Hydrolase Ameliorates
Cisplatin-Induced Nephropathy in Mice [Articles]-
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Authors: Chen, C; Wang, W, Raymond, M, Ahmadinejad, F, Poklis, J. L, Em, B, Gewirtz, D. A, Lichtman, A. H, Li, N.
Pages: 230 - 240
Abstract: Cisplatin is a potent first-line therapy for many solid malignancies, such as breast, ovarian, lung, testicular, and head and neck cancer. However, acute kidney injury (AKI) is a major dose-limiting toxicity in cisplatin therapy, which often hampers the continuation of cisplatin treatment. The endocannabinoid system, consisting of anandamide (AEA) and 2-arachidonoylglycerol and cannabinoid receptors, participates in different kidney diseases. Inhibition of fatty acid amide hydrolase (FAAH), the primary enzyme for the degradation of AEA and AEA-related N-acylethanolamines, elicits anti-inflammatory effects; however, little is known about its role in cisplatin nephrotoxicity. The current study tested the hypothesis that genetic deletion of Faah mitigates cisplatin-induced AKI. Male wild-type C57BL6 (WT) and Faah–/– mice were administered a single dose of intraperitoneal injection of cisplatin (30 mg/kg) and euthanatized 72 hours later. Faah–/– mice showed a reduction of cisplatin-induced blood urea nitrogen, plasma creatinine levels, kidney injury markers, and tubular damage in comparison with WT mice. The renal protection from Faah deletion was associated with enhanced tone of AEA-related N-acylethanolamines (palmitoylethanolamide and oleoylethanolamide), attenuated nuclear factor-B/p65 activity, DNA damage markers p53 and p21, and decreased expression of the inflammatory cytokine interleukin-1β, as well as infiltration of macrophages and leukocytes in the kidneys. Notably, a selective FAAH inhibitor (PF-04457845) did not interfere with or perturb the antitumor effects of cisplatin in two head and neck squamous cell carcinoma cell lines, HN30 and HN12. Our work highlights that FAAH inactivation prevents cisplatin-induced nephrotoxicity in mice and that targeting FAAH could provide a novel strategy to mitigate cisplatin-induced nephrotoxicity.SIGNIFICANCE STATEMENTMice lacking the Faah gene are protected from cisplatin-induced inflammation, DNA damage response, tubular damage, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to mitigate cisplatin-induced nephrotoxicity.
PubDate: 2023-03-16T14:37:28-07:00
DOI: 10.1124/molpharm.122.000618
Issue No: Vol. 103, No. 4 (2023)
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- A Novel Role of Uricosuric Agent Benzbromarone in BK Channel Activation
and Reduction of Airway Smooth Muscle Contraction [Articles]-
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Authors: Gao, J; Yin, H, Dong, Y, Wang, X, Liu, Y, Wang, K.
Pages: 241 - 254
Abstract: The uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K+ channels. Benzbromarone results in dose-dependent activation of macroscopic big potassium (BK) currents about 1.7- to 14.5-fold with an EC50 of 111 μM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Coexpressing β1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 μM and a leftward shift of conductance-voltage (G-V) curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids 329RKK331 in the cytoplasmic linker between S6 and C-terminal regulator of potassium conductance (RCK) gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies.SIGNIFICANCE STATEMENTWe describe a novel role of uricosuric agent benzbromarone in big potassium (BK) channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca2+- and voltage-activated K+ channel (BK channel), which serves numerous cellular functions, including control of smooth muscle contraction. Pharmacological activation of BK channel by the FDA-approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channelopathies.
PubDate: 2023-03-16T14:37:28-07:00
DOI: 10.1124/molpharm.122.000638
Issue No: Vol. 103, No. 4 (2023)
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