Tài liệu Báo cáo khoa học: Specific targeting of a DNA-alkylating reagent to mitochondria Synthesis

Specific targeting of a DNA-alkylating reagent to mitochondria

Synthesis and characterization of [4-((11aS)-7-methoxy-1,2,3,11a-tetrahydro￾5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-8-oxy)butyl]-triphenylphosphonium iodide

Andrew M. James1

, Frances H. Blaikie2

, Robin A. J. Smith2

, Robert N. Lightowlers3

, Paul M. Smith3

and Michael P. Murphy1

1

MRC-Dunn Human Nutrition Unit, Wellcome Trust-MRC Building, Cambridge, UK; 2

Department of Chemistry, University

of Otago, Dunedin, New Zealand; 3

Department of Neurology, Medical School, University of Newcastle upon Tyne, UK

The selective manipulation of the expression and replica￾tion of mitochondrial DNA (mtDNA) within mammalian

cells has proven difficult. In progressing towards this goal

we synthesized a novel mitochondria-targeted DNA￾alkylating reagent. The active alkylating moiety [(11aS)-8-

hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]

[1,4]benzodiazepin-5-one (DC-81)], irreversibly alkylates

guanine bases in DNA (with a preference for AGA tri￾plets), preventing its expression and replication. To target

this compound to mitochondria it was covalently coupled

to the lipophilic triphenylphosphonium (TPP) cation to

form a derivative referred to as mitoDC-81. Incorporation

of this lipophilic cation led to the rapid uptake of

mitoDC-81 by mitochondria, driven by the large mem￾brane potential across the inner membrane. This com￾pound efficiently alkylated isolated supercoiled, relaxed￾circular or linear plasmid DNA and isolated mtDNA.

However mitoDC-81 did not alkylate mtDNA within

isolated mitochondria or cells, even though it accessed the

mitochondrial matrix at concentrations up to 100-fold

higher than those required to alkylate isolated DNA. This

surprising finding suggests that mtDNA within intact

mitochondria may not be accessible to this class of alky￾lating reagent. This inability to alkylate mtDNA in situ

has significant implications for the design of therapies for

mtDNA diseases and for studies on the packaging,

expression and turnover of mtDNA in general.

Keywords: membrane potential; mitochondria; mitochond￾rial DNA; targeting.

Mammalian mitochondrial DNA (mtDNA) encodes 13

polypeptides and the RNA machinery for their transcrip￾tion and translation [1–3]. As these polypeptides are all

components of oxidative phosphorylation complexes,

mtDNA mutations can severely disrupt mitochondrial

function, leading to a number of human diseases for which

there are no effective therapies [2–8]. Possibilities for

treatment, such as the replacement of the defective gene

by gene therapy, are being explored; however, gene therapy

for mtDNA diseases is even more challenging than for

nuclear gene defects because of the problem of delivering

DNA to mitochondria, the difficulty of generating stable

insertion or expression of exogenous DNA within mam￾malian mitochondria, and the large number of mitochon￾dria and mtDNA molecules per cell [9,10]. Even if this

approach is effective, it will not be practical for the majority

of mtDNA diseases that are caused by mtDNA deletions, or

mutations in RNA genes [9,11] until we extend our

knowledge of potential RNA import processes in mamma￾lian mitochondria [12].

Because of these challenges an alternative
antigenomic

strategy has been developed as a potential therapy for

mtDNA diseases [13–16]. This approach does not introduce

a functioning copy of the defective gene; instead it utilizes

the following mtDNA properties: mtDNA is present in

patients at high copy number as a mixture of both normal

and mutated molecules; mtDNA diseases are only pheno￾typically expressed above a threshold proportion of mutated

mtDNA; and mtDNA is continually degraded and resyn￾thesized. Consequently, if the proportion of mutated

mtDNA molecules in a patient can be decreased below this

threshold the disease phenotype may be suppressed. This

could be done by selectively enhancing the degradation, or

inhibiting the replication, of mutated mtDNA molecules

without affecting wild-type mtDNA [17]. The potential of

this approach has been demonstrated for the mtDNA

disease neuropathy, ataxia and retinitis pigmentosa

Correspondence to M. P. Murphy, MRC-Dunn Human Nutrition

Unit, Wellcome Trust-MRC Building, Hills Road,

Cambridge CB2 2XY, UK.

Fax: + 44 1223 252905, Tel.: + 44 1223 252900,

E-mail: [email protected],

http://www.mrc-dunn.cam.ac.uk

Abbreviations: DC-81, (11aS)-8-hydroxy-7-methoxy-1,2,3,11a￾tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one; DMEM,

Dulbecco’s modified Eagle medium; FCCP, carbonylcyanide-p￾trifluoromethoxy-phenylhydrazone; IBTP, 4-iodobutyltriphenyl￾phosphonium iodide; mitoDC-81, [4-((11aS)-7-methoxy-1,2,3,11a￾tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-on-8-oxy)butyl]tri￾phenylphosphonium iodide; mtDNA, mitochondrial DNA; NARP,

neuropathy, ataxia and retinitis pigmentosa; PNA, peptide nucleic

acid; TPMP, methyl triphenylphosphonium; TPP, triphenyl￾phosphonium.

(Received 27 February 2003, revised 15 April 2003,

accepted 12 May 2003)

Eur. J. Biochem. 270, 2827–2836 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03660.x