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Mind Your Ears: A New Antidote to Aminoglycoside Toxicity? Mary E. O’Sullivan and Alan G. Cheng* Stanford University, 801 Welch Road, Palo Alto, California 94305, United States ABSTRACT: Aminoglycoside antibiotics are known toxins to cochlear hair cells, causing permanent hearing loss. Using the zebrafish lateral line system as a platform for drug screen and subsequent validation in the rat cochlea in vivo, Chowdhury et al. characterized a novel otoprotectant working against aminoglycoside-induced hearing loss.
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minoglycoside antibiotics are critical in the treatment of bacterial infections. However, their usage is associated with reversible damage to the kidney (nephrotoxicity) and irreversible damage to the inner ear (ototoxicity), significantly limiting drug administration. As drug dosage and treatment duration correlate with toxicity, monitoring kidney function and serum drug concentrations is used to reduce toxicity. There are currently no co-treatments approved by the U.S. Food and Drug Administration (FDA) or the European Medicines Agency. For affected individuals, hearing aids and cochlear implantation are the only restorative options available once damage has occurred. Around the world, researchers are deploying multiple approaches to tackle this problem. As a strategy, inner ear protection through pharmacological intervention is a long-sought-after goal. In this issue of the Journal of Medicinal Chemistry, Chowdhury et al. report candidate protective compounds that counteract aminoglycoside-induced hearing loss.1 In the inner ear, ototoxicity stems from the death of sensory hair cells which both amplify and convert mechanical inputs derived from sounds into electrical signals that the nervous system can process. Mechanoelectrical transduction takes place when stereocilia at the tips of hair cells are deflected. Deflection triggers opening of mechanoelectrical transduction (MET) channels, stimulating a cascade of cellular events, culminating in the activation of the auditory neurons that relay information centrally. Aminoglycoside entry largely occurs through the MET channel;2 however other mechanisms such as endocytosis can also be involved.3,4 Once inside, aminoglycosides can damage a number of intracellular targets including the endoplasmic reticulum, mitochondrion, and the ribosome (Figure 1) resulting in an anomalous rise in reactive oxygen species and initiating cell death.3,4 The work presented by Chowdhury et al.1 stems from an observation that a thiophene-urea-caboxamide compound, ORC-001, is protective of hair cells from aminoglycosides in the zebrafish lateral line, another hair cell-bearing organ used to detect motion. However, ORC-001 displayed pharmacokinetic and toxicological liabilities including only adequate solubility, moderate oral bioavailability, short in vivo half-life, and the notable side effect of inhibiting the human ether-a-go-go-related gene (hERG) potassium ion channel.1 This channel is linked to cardiac arrhythmias through QT interval prolongation, one of the most common causes of the termination of the use of drugs in the United States.5 To improve the risk/benefit ratio, © XXXX American Chemical Society
Figure 1. Mechanism matters. Aminoglycosides enter outer hair cells through the mechanoelectrical transduction channel (A). Once inside, they interact with many targets including the ribosome (B), the endoplasmic reticulum (C), and the mitochondrion (D). Permission to reprint figure is provided by Alan G. Cheng.
otoprotective potency, and pharmacokinetic properties of ORC-001, Chowdhury et al. characterized a family of ORC001 derivatives and discovered the lead compound 90 (ORC13661) which shows greater otoprotection than ORC-001 with less off-target activity. To achieve this, Chowdhury et al. designed and synthesized over 400 analogues of ORC-001.1 As a strategy for drug design, they conceptualized ORC-001 as six structural units: (1) N3aryl substituents on the urea; (2) the 1,3-disubstituted urea; (3) thiophene core; (4) primary carboxamide C2 substitution; (5) tetrahydropyridine core, and (6) N-ethyl group. They then synthetically modified each of these units and tested derivatives using the external zebrafish lateral line system, a model example of the zebrafish drug screening platform.6 This sensory system, like the mammalian cochlea, is sensitive to aminoglycosides Received: November 7, 2017
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DOI: 10.1021/acs.jmedchem.7b01645 J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
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organelles and signaling pathways. In the search for an otoprotective compound, considerable efforts have focused on mitigating the damaging effects of reactive oxygen species using antioxidants.12 An emerging otoprotective strategy is to limit binding to one of the drugs’ intracellular targets: the ribosome. Aminoglycosides bind to both the cytosolic and mitochondrial ribosomes, and researchers are currently looking to redesign aminoglycoside antibiotics to prevent these drug−target interactions.13−15 Going forward, a greater understanding of compound 90’s otoprotective mechanism may not only aid clinical translation but also facilitate the discovery of additional candidate otoprotectants. Moreover, it may reveal whether this treatment could be protective against other ototoxic agents (e.g., cisplatin). Further testing of the lead compound’s effect on antimicrobial activity will also inform compound 90’s clinical potential. A wider screen of bacterial species and strains, including drug-resistant isolates, could help stratify patients and bacterial infection types during a clinical trial. As research continues, it will be pertinent to determine whether the otoprotective capabilities of compound 90 are long-lasting as aminoglycosides can remain in cochlear hair cells at least for months. Similarly, it will be important to assess whether results in rats are conferred to the guinea pig model, where drug dosing and hearing frequencies are closer to humans. To date, dozens of compounds are reported to have otoprotective function, yet no co-treatment has moved successfully from bench to bedside. The exciting results demonstrated by the current study positions compound 90 as a promising candidate for the prevention of aminoglycosideinduced hearing loss.
with cell death occurring in a dose-dependent fashion. While differences exist between the zebrafish and mammalian systems, the location of the lateral line system on the body surface allows for easy access, thus serving as a powerful high-throughput system capable of screening hundreds of compounds at multiple concentrations. Using the zebrafish model, Chowdhury et al. systematically determined the contribution of each structural element to biological activity. For example, they found that changes to the bis-monosubstituted urea or thiophene-3-carboxamide abolished the compounds’ otoprotective activity whereas addition with the N-ethyltetrahydropyridine moiety was the most fruitful modification. The team moved three novel compounds forward for additional testing. Tests included aqueous solubility, plasma protein binding profiles, elimination kinetics, human hepatic (HepG2) cell line toxicity, oral bioavailability, acute toxicity in vivo, and hERG ion channel inhibition, metrics that will facilitate an FDA Investigational New Drug filling. From this work, compound 90 emerged as the lead compound; it displayed superior protective activity in the zebrafish system, decreased hERG-inhibitory activities, and improved pharmacokinetic properties.1 Remarkably, neither the lead (compound 90) nor the parent (ORC-001) compound interfered with the antimicrobial activities of aminoglycosides in vivo and in vitro. For example, compound 90 exhibited no interference with the in vitro antimicrobial potency of tobramycin, amikacin, kanamycin, and neomycin against five strains of Pseudomonas aeruginosa, a pathogen that is the leading cause of chronic lung infection in patients with cystic fibrosis.7 Compound 90 also showed no interference in an in vivo mouse pulmonary tuberculosis (TB) model with amikacin, and kanamycin−aminoglycosides are critical second line agents in the treatment of multidrug resistant TB.8 In contrast, both the lead and parent compounds were otoprotective in rats in vivo. In the first of two investigations, ORC-001 prevented loss of auditory brain stem responses (ABRs) when rats were administered kanamycin (500 mg kg−1 day−1 which is a supratherapeutic dose designed to induce hearing loss). In the second study, compound 90 prevented hearing loss in rats challenged with amikacin (320 mg kg−1 day−1). Supporting the audiometric data, immunohistochemistry of the inner and outer hair cells of the organ of Corti from experimental animals showed prevention of aminoglycosideinduced hair cell loss by co-treatment with compound 90. Aminoglycoside ototoxicity stems from the death of sensory hair cells, which can occur in a delayed manner because the drug can remain in the inner ear for several months.3,4 Thus, there are many possible mechanisms by which compound 90 exerts its protection, whether that be by preventing antibiotic entry into inner ear fluid spaces and hair cells or by dampening toxicity once inside hair cells (Figure 1). In the past few years, the concept of preventing aminoglycoside entry into hair cells via the MET channel for otoprotection has attracted attention and several compounds have been shown to be protective. For example, inhibiting mechanotransduction by channel blockers, or indirectly by breaking the tip links and by genetic mutation, protects hair cells from aminoglycoside toxicity.2,9,10 Most recently, the MET channel blocker d-tubocurarine (a curare alkaloid) was shown to reduce aminoglycoside loading into rat cochlear hair cells and protective in zebrafish and in vitro murine ototoxicity models.11 Compound 90 may also be protective inside the cell through interactions with different
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Alan G. Cheng: 0000-0002-4702-8401 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank A. Ricci and T. Jan for critical reading and insightful comments and C. Gralapp for figure illustration. REFERENCES
(1) Chowdhury, S.; Owens, K. N.; Herr, R. J.; Jiang, Q.; Chen, X.; Johnson, G.; Groppi, V. E.; Raible, D. W.; Rubel, E. W.; Simon, J. A. Phenotypic optimization of urea-thiophene carboxamides to yield potent, well tolerated and orally active protective agents against aminoglycoside-induced hearing loss. J. Med. Chem. 2017, DOI: 10.1021/acs.jmedchem.7b00932. (2) Alharazneh, A.; Luk, L.; Huth, M.; Monfared, A.; Steyger, P. S.; Cheng, A. G.; Ricci, A. J. Functional hair cell mechanotransducer channels are required for aminoglycoside ototoxicity. PLoS One 2011, 6, e22347. (3) O’Sullivan, M. E.; Perez, A.; Lin, R.; Sajjadi, A.; Ricci, A. J.; Cheng, A. G. Towards the prevention of aminoglycoside-related hearing loss. Front. Cell. Neurosci. 2017, 11, 325. (4) Jiang, M.; Karasawa, T.; Steyger, P. S. Aminoglycoside-induced cochleotoxicity: a review. Front. Cell. Neurosci. 2017, 11, 308. (5) Roden, D. M. Drug-induced prolongation of the QT interval. N. Engl. J. Med. 2004, 350, 1013−1022. (6) Harris, J. A.; Cheng, A. G.; Cunningham, L. L.; MacDonald, G.; Raible, D. W.; Rubel, E. W. Neomycin-induced hair cell death and
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DOI: 10.1021/acs.jmedchem.7b01645 J. Med. Chem. XXXX, XXX, XXX−XXX
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rapid regeneration in the lateral line of zebrafish (Danio rerio). J. Assoc. Res. Otolaryngol 2003, 4, 219−234. (7) Li, Z.; Kosorok, M. R.; Farrell, P. M.; Laxova, A.; West, S. E.; Green, C. G.; Collins, J.; Rock, M. J.; Splaingard, M. L. Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in children with cystic fibrosis. JAMA 2005, 293, 581−588. (8) Guidelines for Treatment of Drug-Susceptible Tuberculosis and Patient Care (2017 Update); WHO: Geneva, Switzerland, 2017. (9) Vu, A. A.; Nadaraja, G. S.; Huth, M. E.; Luk, L.; Kim, J.; Chai, R.; Ricci, A. J.; Cheng, A. G. Integrity and regeneration of mechanotransduction machinery regulate aminoglycoside entry and sensory cell death. PLoS One 2013, 8, e54794. (10) Huth, M. E.; Han, K. H.; Sotoudeh, K.; Hsieh, Y. J.; Effertz, T.; Vu, A. A.; Verhoeven, S.; Hsieh, M. H.; Greenhouse, R.; Cheng, A. G.; Ricci, A. J. Designer aminoglycosides prevent cochlear hair cell loss and hearing loss. J. Clin. Invest. 2015, 125, 583−592. (11) Kirkwood, N. K.; O’Reilly, M.; Derudas, M.; Kenyon, E. J.; Huckvale, R.; van Netten, S. M.; Ward, S. E.; Richardson, G. P.; Kros, C. J. d-Tubocurarine and berbamine: alkaloids that are permeant blockers of the hair cell’s mechano-electrical transducer channel and protect from aminoglycoside toxicity. Front. Cell. Neurosci. 2017, 11, 262. (12) Huth, M.; Ricci, A.; Cheng, A. Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection. Int. J. Otolaryngol. 2011, 2011, 937861. (13) Hobbie, S. N.; Bruell, C. M.; Akshay, S.; Kalapala, S. K.; Shcherbakov, D.; Böttger, E. C. Mitochondrial deafness alleles confer misreading of the genetic code. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3244−3249. (14) Francis, S. P.; Katz, J.; Fanning, K. D.; Harris, K. A.; Nicholas, B. D.; Lacy, M.; Pagana, J.; Agris, P. F.; Shin, J.-B. A novel role of cytosolic protein synthesis inhibition in aminoglycoside ototoxicity. J. Neurosci. 2013, 33, 3079−3093. (15) Matt, T.; Ng, C. L.; Lang, K.; Sha, S.-H.; Akbergenov, R.; Shcherbakov, D.; Meyer, M.; Duscha, S.; Xie, J.; Dubbaka, S. R.; PerezFernandez, D.; Vasella, A.; Ramakrishnan, V.; Schacht, J.; Bottger, E. C. Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 10984−10989.
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DOI: 10.1021/acs.jmedchem.7b01645 J. Med. Chem. XXXX, XXX, XXX−XXX
