Reducing the Toxicity of Designer Aminoglycosides as Nonsense Mutation Readthrough Agents for Therapeutic Targets

Mutation Readthrough Agents for Therapeutic Targets

Letter

Reducing the Toxicity of Designer Aminoglycosides as Nonsense

Michael Popadynec, Alireza Baradaran-Heravi, Benjamin Alford, Scott A. Cameron, Keith Clinch,

Jennifer M. Mason, Phillip M. Rendle, Olga V. Zubkova, Zhonghong Gan, Hui Liu, Oscar Rebollo,

Dennis M. Whit ﬁ eld, Fengyang Yan, Michel Roberge,* and David A. Powell*

Cite This: https://doi.org/10.1021/acsmedchemlett.1c00349

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ABSTRACT: A signiﬁcant proportion of genetic disease cases arise from

truncation of proteins caused by premature termination codons. In eukaryotic

cells some aminoglycosides cause readthrough of premature termination codons

during protein translation. Inducing readthrough of these codons can potentially

be of therapeutic value in the treatment of numerous genetic diseases. A signiﬁcant

drawback to the repeated use of aminoglycosides as treatments is the lack of

balance between their readthrough eﬃcacy and toxicity. The synthesis and

biological testing of designer aminoglycoside compounds is documented herein.

We disclose the implementation of a strategy to reduce cellular toxicity and maintain readthrough activity of a library of compounds

by modiﬁcation of the overall cationic charge of the aminoglycoside scaﬀold through ring I modiﬁcations.

KEYWORDS: Designer aminoglycosides, nonsense mutations, genetic diseases, premature termination codon readthrough

hanges to a single base in the coding region of mRNA

can transform an amino acid codon into a stop codon,

leading to the premature termination of translation. Termed

nonsense mutations, premature termination codons (PTC)

result in the formation of defective, truncated proteins. Genes

are inactivated by nonsense mutations in approximately 11% of

patients across several thousand rare genetic diseases. Addi-

tionally, they also cause tumor suppressor gene inactivation in

−3

about 10% of cancer patients.

Traditionally used as Gram-negative antibacterial agents, the

aminoglycoside class of compounds (Figure 1) have also been

shown to bind and alter the conformation of eukaryotic

ribosomes. This conformational change in the ribosomal A site

allows for the pairing of a near-cognate aminoacyl-tRNA at a

PTC site, inducing readthrough of the codon, and suppressing

premature translation termination. Incorporation of an amino

acid at the PTC can result in the expression of the wild type or

alternative mutant allele and lead to the production of

−6

functional or near-functional proteins.

Harnessing this

Figure 1. Examples of natural aminoglycosides.

mechanism of action has provided an approach to treat

genetic disorders caused by nonsense mutations.

−10

The aminoglycoside class of antibiotics consist of pyranose,

and less commonly furanose, units bound to a central 2-

deoxystreptamine (2-DOS) core. The carbohydrate bound at

position 4 of 2-DOS is ring I, and the carbohydrate bound

either at position 5 or at position 6 of 2-DOS is ring III (rings

labeled in blue, Figure 1). The most clinically useful

aminoglycoside antibiotics are typically disubstituted on the

Received: June 24, 2021

Accepted: August 4, 2021

,5- or 4,6-hydroxy positions of 2-DOS. Natural features

commonly include further glycosidic elongation (3a−b, Figure

XXXX American Chemical Society

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

) and (S)-4-amino-2-hydroxybutanoyl (AHB) substitution at

the ototoxicity of antibacterial agent sisomicin (6a Figure 2)

Letter

the 2-DOS core N-terminals (5, Figure 1).

Aminoglycosides are typically produced as complex mixtures

by fermentation of Gram-positive bacteria, and the challenges

associated with isolating pure components from these mixtures

have further complicated their study.

For example,

gentamicin is composed of gentamicins C1, C1a, C2, and

C2a (1, Figure 1) in addition to various minor components

including sisomicin, gentamicins A, B, B1, X2, 2-DOS,

13−15

garamine, and garosamine.

Therefore, access to pure

synthetic analogs aids in the development of novel amino-

glycosides with favorable therapeutic proﬁles.

Another signiﬁcant challenge with the development of

aminoglycosides as PTC readthrough agents centers around

their clinical toxicity proﬁle. Aminoglycosides, in general,

exhibit nephrotoxic and ototoxic properties. Nephrotoxicity

is often observed early during treatment, while irreversible

ototoxicity incidence increases upon prolonged dosing. The

mechanism of toxicity is attributed to perturbations in various

intracellular process, all driven by accumulation and retention

in the cochlear cells and proximal tubule cells, via

mechanotransducer (MET) channels such as megalin and

8−20

cubulin.

Megalin and cubulin recognize and transport

21−24

highly cationic molecules, including aminoglycosides.

A strategy demonstrated by Ricci and colleagues to lower

Figure 3. Graphical 3D representation (A) and 2D representation (B)

of the key interaction of the four basic amine groups present in G418

,27

(

2) with the yeast 80S ribosome.

The 2′-amino group was

identiﬁed as a potential basic moiety which could be substituted

without detrimentally impacting PTC readthrough activity.

^d^e^t^e^r^mⁱⁿ^e^d^b^y^L^D50 values in mice. The work we present,

herein, will document our eﬀorts to reduce toxicity and

maintain eukaryotic PTC readthrough activity of existing small

molecules by modiﬁcation of the aforementioned amino group.

After initially identifying the paromamine core as a scaﬀold,

several generations of compounds have been reported by

Baasov and colleagues as therapeutically relevant agents

Figure 2. Modiﬁcation of sisomicin to reduce toxicity by Ricci.

was to reduce the cationic charge of the molecule, which was

achieved by eliminating basic sites from the sisomicin skeleton

(

Figure 2). Prior elucidation of the structure of bacterial

ribosome-bound gentamicin C1a (1d, Figure 1) and

paromomycin by X-ray crystallography suggested that the

Ring II 1- and Ring III 3″-amino groups were nonessential sites

for binding, therefore modiﬁcation was not expected to aﬀect

29−35

(Figure 4 ).

Intrigued by the seminal work on designer

therapeutic function. The introduction of amide and

sulfonamide functionality was shown to reduce ototoxicity

and decrease penetration of hair cell MET channels in rat

cochlear cultures, thus providing a strategy to reduce the

toxicity of aminoglycosides for PTC readthrough.

The Ricci et al. study was limited to sisomicin analogues. We

recognized an opportunity to expand on this work by

chemically synthesizing a library of known PTC readthrough

agents with modiﬁed amino groups, hoping to reduce cell

toxicity without compromising readthrough activity.

Examination of the published X-ray crystal structure of the

G418 (2) aminoglycoside bound to the yeast 80S ribosome

suggested that replacement of the Ring I 2′-amino group may

be a suitable starting point for these explorations (Figure

,27

Notably, a direct relationship was previously reported

Figure 4. Evolution of synthetic aminoglycosides developed by

between the basicity of the 2′-amino moiety and toxicity as

Baasov and colleagues as eukaryotic PTC readthrough agents.

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

of cellular readthrough in our DMS-114 system (Figure 6).

Letter

aminoglycosides with improved PTC readthrough activity, we

decided to apply our structural modiﬁcation onto the core of

NB124 (ELX-02), an aminoglycoside derivative in phase II

clinical trials for treatment of cystic ﬁbrosis and nephropathic

readthrough activity. Compounds 9a and 9b have three basic

amine moieties and would possess an overall charge of +3 at

physiological pH. In contrast, NB124 and G418 (2) contain

four basic moieties and a +4 overall charge. If the cationic

nature of the molecule as a whole was important for either

cellular uptake or readthrough activity, then an analogue, such

as compound 9c, may shed some light on the structural drivers

cystinosis.

This led to the design of compound 9a, where the 2′-amino

group was replaced with a hydroxyl moiety (Figure 5). In

Figure 5. First-generation modiﬁed NB124 analogues with 2′-

hydroxyl modiﬁcations.

Figure 6. Second-generation modiﬁed NB124 analogues with 2′-

hydroxyl moieties and 6′/7′ modiﬁcations.

addition to compound (R)-9a, we were able to prepare the

diastereomeric 6′-alcohol, (S)-9b (Figure 5). The impact of

the stereoisomer at the 6′-position on PTC readthrough

activity has not previously been explored, however others have

noted the conformation of the 6′-alcohol impacts antibacterial

The second approach relied on the observation initially

reported by Simonson and colleagues that the introduction of a

hydroxylmethyl group at the ring I 6′,7′-positions in

paromamine did not aﬀect binding to the bacterial ribosome

activity.

To evaluate the PTC readthrough activity of the compounds

we selected the lung carcinoma DMS-114 cell line with

homozygous nonsense mutation R213X in the TP53 gene.

This mutation was previously shown to be very responsive to

relative to the alcohol. Subsequent incorporation of the 7′-

hydroxyl group on NB124 by Baasov and co-workers, however,

suggested improved activity via interactions with RNA bases in

PTC readthrough treatments. DMS-114 cells were exposed

to the compounds at various concentrations over the course of

the eukaryotic ribosome structure (NB157).

This led to the design of compound 9d, which incorporated

the additional primary alcohol at the 7′-position, while also

maintaining the reduced cationic nature of compound 9a

(Figure 6). We were interested in evaluating whether addition

of a 7′-hydroxyl group would be suﬃcient to improve the

readthrough activity to levels similar to that of G418 (2) or

NB124.

As such we sought to synthesize both 9c and 9d to better

understand the structural−activity relationship of charge, ring I

substituents, and PTC readthrough activity.

Similarly to triamine analogue 9b, the tetraamine analogue

9c did not display any PTC readthrough activity at

concentrations up to 500 μg/mL, suggesting that the overall

cationic charge was not essential for readthrough. Most

promisingly, however, was the signiﬁcant concentration

dependent increase in the PTC readthrough activity of the

triamine analogue 9d. This compound induced p53 PTC

readthrough starting at 31 μg/mL and at 125 and 250 μg/mL

readthrough reached levels similar to those elicited by 15 and

31 μg/mL NB124, respectively (Figure 7b). Consistent with

our goal, 9d did not show cell toxicity at concentrations up to

500 μg/mL.

To gain a better understanding of the safety of these novel

compounds, we measured their eﬀect on human ﬁbroblasts

viability and compared them to that of G418 and NB124

(Figure 8). Both novel compounds 9a and 9d did not

signiﬁcantly inhibit cell viability at concentrations up to 0.5

mg/mL, however compound 9d caused a 15% reduction in cell

viability at 1 mg/mL. Both G418 and NB124 caused

signiﬁcant concentration dependent reduction in cell viability

with 1 mg/mL eliciting 72% and 50% reduction, respectively

(Figure 8).

8 hours. Expression of the full-length p53, the PTC

readthrough product, was determined. As anticipated, both

G418 and NB124 induced concentration dependent increase

in PTC readthrough (Figure 7a). NB124 was less active than

G418 at lower concentrations but at 62 μg/mL they both

elicited similar PTC readthrough. NB124 was not toxic to

DMS-114 cells at concentrations up to 62 μg/mL but it

showed signiﬁcant toxicity at 125 μg/mL and higher

concentrations. Replacement of the 2′-amine moiety with the

alcohol led to a signiﬁcant reduction in the PTC readthrough

activity of compounds 9a and 9b compared to parent molecule

NB124 (Figure 7a). We observed a concentration dependent

increase in the production of full-length p53 with 9a starting at

25 μg/mL. At 500 μg/mL, this compound elicited PTC

readthrough that reached 18% of the maximal PTC read-

through activity of the 62 μg/mL nontoxic dose of NB124

(

Figure 7a). Compound 9b did not show any PTC

readthrough activity at concentrations up to 500 μg/mL

indicating the importance of the stereoisomer at the 6′-

position for PTC readthrough activity (Figure 7a). In addition

to decreased PTC readthrough activity, we also observed a

marked reduction in cytotoxicity of 9a and 9b at concen-

trations up to 500 μg/mL compared to G418 or NB124.

Despite the reduction in activity, a comparable toxicity versus

eﬃcacy value for compound 9a, encouraged us to further

explore the structure−activity relationship. Two approaches

were considered to further understand and improve the

structural aspects driving the PTC readthrough activity of

these aminoglycoside derivatives.

The ﬁrst was to understand whether the overall cationic

charge of the molecule was important for driving cellular

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

against vinculin, relative to that of NB124 at 62 μ g/mL.

Letter

groups and conversion to the corresponding sulfate salts

yielded the ﬁnal products for biological evaluation.

The ring I analogues of compounds 9a and 9b were

prepared in an almost-identical fashion (Scheme 2). Thio-

glycosides 10a−b were oxidized at the 6′-positions to aﬀord

0,41

aldehydes 11a−b.

Treatment with methylmagnesium

bromide gave alcohols 12a−b as diastereomeric mixtures.

Benzoylation aﬀorded diastereomerically pure glycosyl donors

3a−b after chromatographic separation, in preparation for

coupling to ring II.

Scheme 2

Figure 7. Eﬀect of compounds 9a−b (A) and 9c−d (B) on TP53

PCT readthrough. DMS-114 were exposed to various concentrations

of indicated compounds for 48 h, and p53 levels (full-length, FL-p53;

truncated, TR-p53) were determined using capillary electrophoresis

western analysis. Vinculin was used as loading control. The numbers

under the lanes indicate the percent amounts of FL-p53 normalized

0−12a: R = Tol, R = OH, R = Me. 10−12b: R = Ph, R = Me,

R = OH. Route towards 13a−b: (a) DMP, CH Cl ; (b) MeMgBr,

THF; (c) BzCl, pyridine.

The ring I synthesis of analogue 9c began with known azide

4, accessed stereoselectively from L-ascorbic acid (Scheme

). Global acyl protection enabled the β-thioglycoside 15 to

Scheme 3

Figure 8. Fibroblast toxicity studies on 9a and 9d. Wild-type

ﬁbroblasts were exposed to various concentrations of indicated

compounds for 48 h and cell viability was measured using the

Promega ApoLive-Glo Multiplex Assay kit.

The synthesis of compounds 9a−d was achieved by coupling

ring I to ring II via chemical glycosylation (Scheme 1). The

intermediate was subsequently coupled to ring III to give the

desired aminoglycoside skeleton. Cleavage of the protecting

Route towards 13c: (a) Ac O, NaOAc; (b) TolSH, BF ·OEt ,

CH Cl ; (c) NaOMe, MeOH then Amberlyte 15 (H ); (d) BnBr,

NaH, DMF.

Scheme 1. Generalized Synthetic Strategy Towards

Aminoglycosides 9a−d

subsequently be accessed selectively over two steps. Further

protecting group manipulations aﬀorded ring I (13c) ready for

coupling to ring II.

The synthesis of analogue 9d began from the tetrabenzylated

glucose species 16 (Scheme 4). Oxidation of 16 under Swern

conditions followed by Wittig methylenation elongated the

ring I framework to give 17. The resulting vinyl group

underwent Upjohn dihydroxylation to give the corresponding

glycol as a diastereomeric mixture. Following stannylene

acetal-mediated selective protection of the primary 7′-alcohol,

the desired stereoisomer 18 was isolated after chromatographic

separation. X-ray crystallography conﬁrmed the stereo-

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

The Supporting Information is available free of charge at

Letter

Scheme 4

Scheme 5

Route towards 13d: (a) (COCl) , DMSO, NEt , CH Cl ; (b)

MePPh Br, NaHMDS, THF; (c) OsO , NMO, tBuOH/acetone/

H O; (d) Bu SnO, MeOH then BnBr, TBABr, toluene; (e) BnBr,

NaH, DMF; (f) Ac O, H SO , AcOH.

3/20−22a: R = OBz, R = Me, R = STol. 13/20−22b: R = Me,

chemistry of 18. Benzyl protection of 18 gave fully benzylated

species 19. Treatment with acetic anhydride in the presence of

sulfuric acid displaced the 1′- and 7′-benzyl groups selectively

R = OBz, R = SPh. 13/20−22c: R = N , R = Me, R = STol. 13/

9b: R = Me, R = OH. 9c: R = NH , R = Me. 9d: R = OH, R =

0−22d: R = OBn, R = CH OAc, R = OAc. 9a: R = OH, R = Me.

1 2 1 2 1 2

to aﬀord diacetate 13d as the glycosyl donor.

OH. Route towards 9a−d: (a) 23; for 13a−c plus NIS, TfOH,

CH Cl /Et O; for 13d plus TMSOTf, 4 Å mol. sieves, CH Cl /Et O;

Thioglycosides 13a−c were treated with NIS in the presence

(

b) NaOMe, THF/MeOH; (c) Ac O, pyridine; (d) 24, BF ·OEt , 4

of triﬂic acid and underwent glycosylation reactions with

Å mol. sieves, CH Cl ; (e) NaOMe, MeOH; (f) not applied to 22a−

35,45

alcohol 23, which was accessed enzymatically (Scheme 5).

b; for 22c−d PMe , NaOH, THF/water; (g) for 22a−b hydrazine

Acetate 13d was reacted with 23 in the presence of triﬂic acid.

Global acetate cleavage of 20a−d, followed by regioselective

monohydrate, Pd/C, MeOH; for 22c−d H , Pd/C, AcOH/H O; (h)

H SO (aq.). TCA = trichloroacetimidate.

46,47

reacylation, aﬀorded alcohols 21a−b as glycosyl acceptors.

A glycosylation reaction with known glycosyl donor trichlor-

ASSOCIATED CONTENT

32,48

■

oacetimidate ring III (24)

was carried out under standard

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00349.

Lewis acidic conditions to give the ﬁnal protected amino-

glycosides (22a−d). Cleavage of the protecting groups,

followed by treatment with sulfuric acid, gave the correspond-

ing sulfate salts 9a−d for biological evaluation.

Experimental procedures toward the synthesis of

compounds 9a−d and associated characterization data,

a description of the biological evaluation procedures,

and X-ray crystallography data (PDF)

We have prepared a series of structural variants of NB124

and NB157, which have illuminated the role of the amino- and

hydroxyl-groups at the 6′- and 2′-position of ring I. The goal of

these synthetic eﬀorts was to reduce the overall cationic charge

of the parent molecules in an attempt to widen the therapeutic

window for readthrough activity over dose-limiting nephrotox-

icity and ototoxicity. PTC readthrough agents exhibit the

potential to treat a wide range of severe genetic diseases,

however the safety proﬁle of aminoglycoside readthrough

agents will need to be improved upon to maximize the

therapeutic potential. Highly cationic charged aminoglycosides

are taken up into proximal tubule and cochlear cells via

transporters such as megalin, which recognize cationic

molecules. Reductions in the overall cationic nature of the

aminoglycosides may serve to reduce overall toxicity but must

not come at the expense of readthrough activity. Our eﬀorts to

identify potent aminoglycoside agents with reduced overall

cationic charge resulted in the identiﬁcation of compound 9d

as a promising analogue. Further evaluation of the in vivo

properties and readthrough activity of compound 9d, together

with assessment of the toxicity proﬁle remains ongoing.

AUTHOR INFORMATION

■

Corresponding Authors

Michel Roberge − Department of Biochemistry and Molecular

Biology, University of British Columbia, Vancouver, British

Columbia V6T 1Z3, Canada; Email: michelr@mail.ubc.ca

David A. Powell − Inception Sciences Canada, Vancouver,

British Columbia V5T 4T5, Canada; Present

Address: Chinook Therapeutics, Inc., Vancouver, BC;

orcid.org/0000-0002-8710-7390; Email: dpowell@

chinooktx.com

Authors

Michael Popadynec − Ferrier Research Institute, Victoria

University of Wellington, Wellington 6012, New Zealand

Alireza Baradaran-Heravi − Department of Biochemistry and

Molecular Biology, University of British Columbia,

Vancouver, British Columbia V6T 1Z3, Canada

Benjamin Alford − Ferrier Research Institute, Victoria

University of Wellington, Wellington 6012, New Zealand

ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters

orcid.org/0000-0003-2669-8622

Letter

Scott A. Cameron − Ferrier Research Institute, Victoria

University of Wellington, Wellington 6012, New Zealand;

Evans, M. J.; Sorscher, E. J.; Bedwell, D. M. Aminoglycoside

suppression of a premature stop mutation in a Cftr −/− mouse

carrying a human CFTR-G542X transgene. J. Mol. Med. 2002, 80,

(

95−604.

Keith Clinch − Ferrier Research Institute, Victoria University

of Wellington, Wellington 6012, New Zealand

Jennifer M. Mason − Ferrier Research Institute, Victoria

University of Wellington, Wellington 6012, New Zealand

Phillip M. Rendle − Ferrier Research Institute, Victoria

University of Wellington, Wellington 6012, New Zealand

Olga V. Zubkova − Ferrier Research Institute, Victoria

University of Wellington, Wellington 6012, New Zealand;

orcid.org/0000-0002-6892-8812

Zhonghong Gan − Sussex Research Laboratories, Inc.,

Ottawa, Ontario K1A 0R6, Canada

Hui Liu − Sussex Research Laboratories, Inc., Ottawa, Ontario

K1A 0R6, Canada

Oscar Rebollo − Sussex Research Laboratories, Inc., Ottawa,

Ontario K1A 0R6, Canada

Dennis M. Whitﬁeld − Sussex Research Laboratories, Inc.,

Ottawa, Ontario K1A 0R6, Canada

Fengyang Yan − Sussex Research Laboratories, Inc., Ottawa,

Ontario K1A 0R6, Canada

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thiazole-4-carboxamides enhance readthrough of premature termi-

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acsmedchemlett.1c00349

9) Manuvakhova, M.; Keeling, K. I. M.; Bedwell, D. M.

Aminoglycoside antibiotics mediate context-dependent suppression

of termination codons in a mammalian translation system. RNA 2000,

Author Contributions

(

, 1044−1055.

D.A.P., A.B.H., and M.R. conceived and designed all

compounds for biological evaluation. The syntheses of all

compounds were designed, performed and characterized by

B.A., K.C., Z.G., J.M.M., O.R., D.M.W., F.Y., H.L., O.V.Z., and

P.M.R. D.A.P., A.B.H., and M.R. designed and performed

biological evaluations of ﬁnal compounds. S.A.C. obtained X-

ray crystallography data. M.P. wrote the manuscript with

contributions from D.A.P., A.B.H., and M.R. M.P. and A.B.H.

compiled the Supporting Information. All authors have given

approval to the ﬁnal version of the manuscript.

10) Clancy, J. P.; Bebök, Z.; Ruiz, F.; King, C.; Jones, J.; Walker, L.;

Greer, H.; Hong, J.; Wing, L.; Macaluso, M.; Lyrene, R.; Sorscher, E.

J.; Bedwell, D. M. Evidence that systemic gentamicin suppresses

premature stop mutations in patients with cystic fibrosis. Am. J. Respir.

Crit. Care Med. 2001, 163, 1683−1692.

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glycosides: the race to develop improved antibiotics and compounds

for the treatment of human genetic diseases. Org. Biomol. Chem. 2008,

6, 227−239.

(12) Mingeot-Leclercq, M.-P.; Glupczynski, Y.; Tulkens, P. M.

Aminoglycosides: activity and resistance. Antimicrob. Agents Chemo-

ther. 1999, 43, 727−737.

Funding

Seed funding for this work was provided by Versant Ventures.

(13) Vydrin, A. F.; Shikhaleev, I. V.; Makhortov, V. L.; Shcherenko,

Notes

N. N.; Kolchanova, N. V. Component composition of gentamicin

The authors declare the following competing ﬁnancial

interest(s): D.A.P. is a former employee of Inception Sciences

Canada and is a current employee of Chinook Therapeutics,

Inc., and owns stock and/or holds stock options in the

Company.

sulfate preparations. Pharm. Chem. J. 2003, 37, 448−450.

(14) Friesen, W. J.; Johnson, B.; Sierra, J.; Zhuo, J.; Vazirani, P.; Xue,

X.; Tomizawa, Y.; Baiazitov, R.; Morrill, C.; Ren, H.; Babu, S.; Moon,

Y.-C.; Branstrom, A.; Mollin, A.; Hedrick, J.; Sheedy, J.; Elfring, G.;

Weetall, M.; Colacino, J. M.; Welch, E. M.; Peltz, S. W. The minor

gentamicin complex component, X2, is a potent premature stop

codon readthrough molecule with therapeutic potential. PLoS One

2018, 13, No. e0206158.

ABBREVIATIONS

■

-DOS, 2-deoxystreptamine; AHB, (S)-4-amino-2-hydroxybu-

tanoyl; DMF, dimethylformamide; DMP, Dess−Martin

periodinane; DMSO, dimethyl sulfoxide; EC , eﬀective

(15) Rajasekaran, P.; Crich, D. Synthesis of gentamicin minor

components: gentamicin B1 and gentamicin X2. Org. Lett. 2020, 22,

3850−3854.

(16) Mingeot-Leclercq, M.-P.; Tulkens, P. M. Aminoglycosides:

concentration, 50%; LC , lethal concentration, 50%; MET,

nephrotoxicity. Antimicrob. Agents Chemother. 1999 , 43 , 1003 −1012.

17) Xie, J.; Talaska, A. E.; Schacht, J. New developments in

aminoglycoside therapy and ototoxicity. Hear. Res. 2011, 281, 28−37.

18) Quiros, Y.; Vicente-Vicente, L.; Morales, A. I.; Lopez-Novoa, J.

mechanotransducer; NaHMDS, sodium hexamethyldisilazide;

NIS, N-iodosuccinimide; NMD, nonsense-mediated mRNA

decay; NMO, N-methylmorpholine N-oxide; OTCA, trichlor-

oacetimidate; PTC, premature termination codon; TCA,

trichloroacetonitrile; THF, tetrahydrofuran

M.; Lopez-Hernandez, F. J. An integrative overview on the

mechanisms underlying the renal tubular cytotoxicity of gentamicin.

Toxicol. Sci. 2011, 119, 245−256.

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