The challenge of developing robust drugs to overcome resistance

Drug Discovery Today � Volume 16, Numbers 17/18 � September 2011 PERSPECTIVE

feature
The challenge of developing robustdrugs to overcome resistance
Amy C. Anderson1,2, Michael P. Pollastri1,3, Celia A. Schiffer1,4,*, [email protected] and

Norton P. Peet1,5, [email protected]

s�PERSPECTIVE

Drug resistance is problematic in microbial disease, viral disease and cancer. Understanding at the outset

that resistance will impact the effectiveness of any new drug that is developed for these disease categories

is imperative. In this Feature, we detail approaches that have been taken with selected drug targets to

reduce the susceptibility of new drugs to resistance mechanisms. We will also define the concepts of

robust drugs and resilient targets, and discuss how the design of robust drugs and the selection of

resilient targets can lead to successful strategies for combating resistance.

Feature
Introduction
Drug resistance is defined, in a clinical setting, as

the point at which administration of the drug can

no longer safely treat the disease state owing to

an induced change in the drug target or an

inability of the drug to reach the target. With an

antimicrobial agent, clinical resistance occurs

when the minimum inhibitory concentration

(MIC) of the drug, for a given microbial strain,

exceeds the concentration of drug that can safely

be administered. Resistance to a drug canarise by:

(i) mutation of the gene (or gene cluster); (ii)

acquisition of extrachromosomal DNA, or a

transposable plasmid, that carries the resistance

gene or genes; (iii) upregulation of the target or;

(iv) upregulation of an efflux mechanism.

We have recently suggested [1] an approach

for combating drug resistance which involves

the selection of resilient drug targets [2] that are

evolutionally constrained and the development

of robust drugs [3] that are less susceptible to the

development of resistance.

A drug target is likely to develop resistance if it

cannot easily tolerate change and maintain

1359-6446/06/$ - see front matter � 2011 Elsevier Ltd. All rights reserv

function. Many drug targets are enzymes.

Because these enzymes perform highly con-

strained and crucial chemical reactions, they can

represent resilient targets that are less suscep-

tible to drug resistance. However, disrupting the

therapeutic target activity is necessary but not

sufficient for developing a drug that avoids

resistance.

A robust inhibitor is one that successfully

inhibits a resilient target, and one that does not

lose effectiveness quickly owing to resistance.

Such an inhibitor could only bind to crucial

regions within the target that would be essential

for function and, thus, intolerant to change. The

use of high-resolution structures and evolu-

tionary constraints aids the design of robust

inhibitors. By choosing resilient targets and

designing robust inhibitors the Institute for Drug

Resistance (IDR) [1] proposes to focus on drug

resistance in drug design strategies, and develop

a new generation of more-effective therapeutics.

This Feature will focus on selected, potentially

resilient drug targets and describe efforts to

produce drugs with various degrees of robust-

ed. doi:10.1016/j.drudis.2011.07.001

ness for these targets in the disease areas of

cancer and bacterial and viral infections. The

highlighted drug targets include: BCR-ABL

kinase; epithelial growth factor receptor (EGFR)

kinase and platelet-derived growth factor

(PDGFR) kinase in cancer; dihydrofolate reduc-

tase (DHFR) and DNA gyrase/topoisomerase IV in

bacteria; and human immunodeficiency virus

(HIV) protease and hepatitis C (NS3/4A) protease

in viruses. Because enzymes are evolutionally

constrained to catalyze a chemical reaction, they

have the potential of being resilient targets;

however, properly identifying robust inhibitors

remains a challenge for the field.

Drug resistance in enzyme targets of

cancer

Drug resistance is now a widespread problem in

cancer and is particularly problematic with kinase

inhibitors, proteasome inhibitors [4,5] and

monoclonal antibodies [6]. Of these, kinase inhi-

bitors comprise the largest class of anticancer

agents where drug resistance has significantly

limited treatment. In this article the focus will be

www.drugdiscoverytoday.com 755

http://dx.doi.org/10.1016/j.drudis.2011.07.001

PERSPECTIVE Drug Discovery Today � Volume 16, Numbers 17/18 � September 2011

Features

�PERSPECTIVE

on the challenges relating to developing robust

drugs that overcome resistance to inhibitors of

the kinase domains of BCR-ABL, EGFR and PDGF.

BCR-ABL inhibitors

Gleevec1 (imatinib) shows remarkable efficacy

by achieving clinical remission in chronic mye-

loid leukemia (CML). Imatinib inhibits the con-

stitutively active kinase domain, c-Abl, of the

fusion protein BCR-ABL [7] existing in CML

patients. Evidence from biochemical and crys-

tallographic studies [8] shows that imatinib

selectively binds a unique conformation of the

activation loop in which a conserved phenyla-

lanine, a member of the Asp-Phe-Gly (DFG) trio

of amino acids, undergoes an extensive 10 A

conformational change, into what is called the

DFG-out conformation [9,10]. Imatinib binding

maintains the kinase in the inactivated state.

Interestingly, many kinases cannot adopt an

inactive conformation [11]; therefore, targeting

this state yields inhibitors with selectivity over

other kinases. Crystal structures of other kinases

in the DGF-out conformation include: KIT (mast/

stem cell growth factor receptor); Aurora A; EGFR

(covered in this article); p38 or mitogen-acti-

vated protein kinase (MAPK)14; kinase insert

domain receptor (KDR); and BRAF [9].

However, the success of imatinib was limited

because of the selection of resistant mutants.

Specifically, the BCR-ABL variant with a T315I

gatekeeper mutation in the ATP-binding site was

observed in 10–20% of CML patients after failure

of imatinib [12,13]. Consequently, there are�200follow-on inhibitors of BCR-ABL kinase [14–16].

Unfortunately, it has been difficult to develop

robust inhibitors against the mutant variant of

BCR-ABL. Two recently approved agents, dasati-

nib and nilotinib (Fig. 1a), have shown success

against imatinib-resistant CML in clinical trials.

However, the BCR-ABL1 T315I variant also confers

resistance to nilotinib and dasatanib [17–21].

Thus, the problem of resistance to this class of

inhibitors is still unsolved. Approaches that are

being taken to develop robust drugs that are

effective against resistant BCR-ABL include: (i) the

design of compounds to inhibit the mutated

enzyme; (ii) allosteric inhibitors that bind at a site

different from the ATP-binding site; (iii) inhibitors

that target other tyrosine kinases and simulta-

neously inhibit the T315I variant of BCR-ABL; and

(iv) development of other dual or multikinase

inhibitors that simultaneously inhibit the ABL and

SFK (SRC family of kinases) families [15]. The SFKs

are thought to be involved in the proliferation of

BCR-ABL1-expressing cell lines [15,16,22]. The

outcomes of these approaches will be crucial to

the successful management of CML.

756 www.drugdiscoverytoday.com

EGFR inhibitors

Because EGFR, a receptor tyrosine kinase, plays a

crucial part in cellular signaling leading to

growth, proliferation and metastasis [23–30],

some investigators believed that inhibitors of its

function would serve as leads for antineoplastic

agents. Gefitinib (Fig. 1b) was approved in 2003

for the treatment of non-small-cell lung cancer

(NSCLC) after failure of platinum-based and

docetaxel therapies, but did not significantly

improve survival [31]. In 2004 erlotinib (Fig. 1b)

was also approved for NSCLC (after failure of one

other agent) and approved in 2005 for pan-

creatic cancer in combination with gemcitabine.

Both compounds are anilinoquinazolines that

compete with ATP to bind the active site of the

EGFR tyrosine kinase; a cocrystal structure of

erlotinib bound to the EGFR tyrosine kinase [32]

reveals the details of that interaction (Fig. 2a).

Genetic evidence shows that resistance occurs

in response to gefinitib and erlotinib in EGFR

through mutation of the ‘gatekeeper’ residue

T790M [33,34], similar to mechanisms of resis-

tance that occur with mutants T315I (BCR-ABL/

imatinib [35]), T6741 (PDGFR/imatinib [36]) and

T670I (c-KIT/imatinib [37]). Interestingly, there is

evidence that the T790M mutation exists before

the initiation of therapy [34] and that cells with

the mutation are then selected during therapy.

The T790M mutation was thought to introduce

steric bulk that interfered with the binding of the

inhibitors [38]. However, crystal structures and

biochemical experiments with the EGFR T790M

and T790M/L858R variants bound to inhibitors

reveal that the T790M mutation increases ATP

affinity, especially in the context of the L858R

mutation that activates the kinase [39]. By

increasing affinity for ATP, the T790Mmutation is

considered to be a multi-drug resistant mutation

that will reduce the potency of any ATP-com-

petitive inhibitor.

The development of irreversible inhibitors

such as HKI272 (Figs 1b and 2b), which is now in

Phase II trials [33,40,41], can overcome the

effects of the T790M mutation. The irreversible

inhibitors have a Michael acceptor that forms a

covalent bond with a cysteine thiol in the active

site pocket, thus eliminating the equilibrium

condition with ATP. HKI272 has been shown to

inhibit EGFR function even in the presence of the

T790M mutation, but could be subject to resis-

tance via a C797 mutation [40].

PDGFR inhibitors

PDGFR tyrosine kinase inhibitors have been

investigated as antineoplastic agents that inhibit

angiogenesis. PDGFR inhibitors are also crucial in

treating hypereosinophilic syndrome (HES), a

disease inwhich the patient has a prolonged state

of eosinophilia that can lead to organ dysfunction

and death [36]. Some patients with this disease

respond favorably to treatment with imatinib,

because they harbor a fused Fip1-like 1 (FIP1L1)-

PDGFRa gene that generates an activated kinase.

After initial treatment with imatinib, resistance is

once again selected with the mutation of the

gatekeeper residue T674I in the kinase domain of

PDGFR. Interestingly, in an analogous fashion as

EGFR, the gatekeeper mutation in many kinases,

including PDGFR, does not exert a steric effect but

has a role in favoring an activated kinase [42].

Several compounds in different stages of devel-

opment are successful at overcoming the T674I

variant (Fig. 1c). Ki11502 [43] is a potent multi-

kinase inhibitor of PDGFRa/b and inhibits T674I as

well as KIT, KDR and FLT3. Nilotinib [44] and

EXEL0862 [45] also inhibit the growth of cells

transfected with FIP1L1-PDGFRa harboring the

T674I mutation. PKC412 (midostaurin) [36], a

natural product isolated from Streptomyces, is

reported to be an effective treatment for FIP1L1-

PDGFRa-induced disease and of imatinib-

induced resistance. Although it is still ATP-com-

petitive, PKC412 could have a different binding

mode than imatinib, making it less susceptible to

imatinib-specific resistance. Sorafenib is an inhi-

bitor of the RAF-1, B-RAF, vascular endothelial

growth factor receptor (VEGFR) and PDGFR tyr-

osine kinases and is also active against cells

harboring the T674I mutation [46]. The inhibitor

HG78501 is reported to bind kinases with the

T674I mutation [47]. A cocrystal structure of

HG78501 with Src kinase shows that the com-

pound forms a H-bond with a neighboring

methionine (Met 341 in Src), possibly explaining

how HG78501 overcomes the effects of the

mutation.

Drug resistance in enzyme targets of

antibiotics

Antibacterial agents, like antineoplastic agents,

target fast-growing proliferative cells. Although

the targets differ, the occurrence of resistance is

common to both classes of drugs and the

development of robust drugs to overcome

resistant variants is an active area of research. In

this article, we highlight efforts to develop

robust drugs against DHFR and gyrase/topoi-

somerase IV.

Dihydrofolate reductase and trimethoprim

resistance

Trimethoprim (TMP) is a potent inhibitor of

bacterial species with the essential enzyme

DHFR, which plays a crucial part in the folate

biosynthetic pathway. TMP, coadministered with


[(Figure_1)TD$FIG]

(a)

(b)

(c)

(d)

(e)

A B C

Imatinib (Gleevec) Nilotinib

Dasitinib (Sprycel)

Gefitinib

Ki11502

Nilotinib

PCK412 (Midostaurin)

Sorafenib

Nalidixic acid Ciprofloxacin Moxifloxacin GSK299423

PfizerVertex

HG78501

Erlotinib

HKl272

Drug Discovery Today

FIGURE 1

(a) Structures of first- and second-generation BCR-ABL kinase inhibitors. (b) Structures of EGFR tyrosine kinase inhibitors. (c) Structures of PDGFR inhibitors. (d)Inhibitors that target the GyrA/ParA subunit of gyrase and/or TopoIV. (e) Benzimidazole GyrB/ParE inhibitors.


Features�PERSPECTIVE


[(Figure_2)TD$FIG]

(a) (b)

Cys 797

HKl272T290M


FIGURE 2

Crystal structures of EGFR (green) with bound inhibitors (purple ormagenta). (a) Erlotinib competes with ATP in the active site of EGFR; (b)HKI272 forms a covalent

link with Cys 797 to eliminate the equilibrium between inhibitor and ATP.


FIGURE 3

Crystal structure of moxifloxicin bound to GyrA [59].

Features

�PERSPECTIVE

sulfamethoxazole (SMZ), is an effective thera-

peutic agent used to treat community-acquired

methicillin-resistant Staphylococcus aureus (CA-

MRSA). However, epidemiological surveys show

that �28% of MRSA strains are TMP-resistant

[48–50]. A common resistance mechanism

involves the acquisition of one of two sets of

chromosomal mutations in the DHFR gene,

either H30N/F98Y or F98Y/H149R. Structures of

wild-type and mutant (F98Y and H30N/F98Y) S.

aureus DHFR with several novel propargyl-linked

antifolates show that the structural effects of the

resistance mutations are subtle [51,52].

Development of compounds that have more

interactions within the DHFR active site has been

the predominant strategy to overcome TMP

resistance. Iclaprim, which underwent Phase III

trials but did not continue thereafter in devel-

opment, achieved nanomolar affinity for the

wild-type and mutant enzymes and showed a

lower propensity for resistance [53]. Crystal

structures of iclaprim bound to the wild-type and

mutant enzymes show additional hydrophobic

interactions in a highly conserved area of the

substrate-binding site [54]. Reported propargyl-

based inhibitors also interact more substantially

with the active site [51,52] than TMP and are less

affected by the mutations that cause TMP

resistance; further experiments to assess their

propensity for resistance are ongoing.

DNA gyrase and topoisomerase IV

inhibitors

In Gram-negative and Gram-positive bacteria

gyrase and topoisomerase IV, respectively, are

important drug targets [55] that serve parallel

functions and are often targeted with the same

compounds. To perform the processes of tran-

scription, replication, repair and recombination,


topoisomerases catalyze either a transient sin-

gle-strand break (Type I) or a double-strand

break (Type II) in supercoiled DNA. In bacteria,

gyrase introduces negative supercoils for the

entire chromosome, creating condensed

packages of genetic material that can be divided

correctly during cell division [56,57].

Fluoroquinolones are an established class of

drugs (Fig. 1d) that stabilize the covalent DNA-

gyrase complex and thehomologousDNA-topoIV

complex. The prototypical quinolone antibiotic

nalidixic acid was discovered in the early 1960s

and has since been replaced by compounds with

higher potency and improved dosing modalities

such as moxifloxicin and ciprofloxacin.

Resistance has emerged to the quinolone

antibiotics in Gram-positive and Gram-negative

strains. A summary of hospital surveys from 1997[(Figure_3)TD$FIG]

to 2001 showed an expanding rate of resistance

across infectious species [58]. Resistance

mechanisms include increasing drug efflux or

mutating the genes that code for DNA gyrase

and topoisomerase IV. Structural studies of a

DNA–topoIV cleavage complex with bound

moxifloxicin (Fig. 3) reveal the effects of amino

acid mutations in the quinolone-resistance-

determining region (QRDR), located in the GyrA

subunit [59]. Other drug-binding models

attempt to explain the effects of other resis-

tance-conferring mutations [60,61]. Compounds

that inhibit DNA cleavage via a new binding

mechanism that evades the fluoroquinolone

resistance mutations and compounds that

simultaneously inhibit GyrB/ParE ATPase func-

tion represent promising new approaches to

overcome resistance.


[(Figure_4)TD$FIG]


FIGURE 4

Crystal structure of GSK299423 bound to ParB [62].


The topoIV/GyrA subunit inhibitor GSK299423

(Fig. 4) inhibits the turnover of the DNA cleaved

complex by stabilizing the enzyme–DNA complex

before cleavage and inhibiting separation of the

DNA strands. In this way, GSK299423 employs a

mechanism that is significantly different from that

of the fluoroquinolones [62]. The binding site for

GSK299423 is highly conserved in bacteria and

does not overlapwith the known binding sites for

fluoroquinolones, creating a potent inhibitor that

shows antibacterial activity against a range of

resistant strainswithmutations inDNAgyrase and

topoisomerase IV.

Using the topology of the binding sites of DNA

gyrase (GyrB) and topoisomerase IV (ParE),

(reviewed in Ref. [63]), novel classes of inhibitors

of the ATPase activity have been designed to

circumvent the resistance observed with qui-

nolones. Interestingly, these compounds also

overcome the resistance associated with the

coumarins and cyclothialidines [64,65] that bind

in a region that overlaps the ATP-binding site

[66,67]. These ATPase inhibitors inhibit gyrase

and topoisomerase IV simultaneously, thus

yielding an advantage in that the emergence of

resistance would require the unlikely occurrence

of simultaneous mutations. Combinations of

virtual screening and HTS led to compound A

(Fig. 1e), which was of modest potency. Com-

pound A was optimized using structure-based

design to arrive at B, a compound that displayed

nanomolar Ki values against S. aureus and

Escherichia coli gyrase and topoisomerase IV, and

impressive MIC90 values of 0.03–0.12 mg/ml

against a range of bacteria. This compound has

since spawned a wide variety of related che-

motypes from several other companies [65,68–

70], such as compound C, recently reported by

Pfizer [69].

Drug resistance in enzymes targeted by

antiviral agents

Most antiviral therapy is rightly focused on vac-

cine development. However, for a growing

number of viruses vaccines have remained elu-

sive, or the virus evolves so quickly that their

scope is limited. Although some successful anti-

viral agents target viral entry, several target the

viral enzymes that include: proteases, transcrip-

tases, integrases and neuraminidases. As with all

quickly evolving diseases there are challenges in

developing inhibitors that are robust; one strategy

is to target enzymes that are evolutionarily con-

strained. The viral proteases, which cleave several

diverse substrates, represent anexample of sucha

class and, to retain activity, the protease must

cleave all the substrates.

HIV protease inhibitors

HIV protease, which cleaves Gag and Gag-Pro-Pol

polyproteins at ten varied sites necessary for the

maturation of virus [71], is a major therapeutic

target for antiviral drugs. During the past 20

years, structure-based drug discovery efforts

have led to the development of nine approved

competitive active site protease inhibitors (PIs).

These inhibitors are the most potent anti-HIV

drugs and essential components of highly active

antiretroviral therapy (HAART) [72,73].

The development of drug resistance is a major

reason for the failure of PI therapy. The virus

accumulates many mutations within the pro-

tease that prevent PIs from binding to the pro-

tease. More than half the residues within the

protease mutate in different combinations and

lead to drug resistance [74,75]. Drug resistance is

a subtle change in the balance of recognition

events: the protease is still able to recognize and

process the natural substrate sites in the Gag and

Gag-Pro-Pol polyprotein, at the same time no

longer being effectively inhibited by the com-

petitive inhibitor. When resistance emerges the

interactions of the protease with an inhibitor are

significantly altered, whereas the interactions

with a natural substrate are maintained.

Crystallographic studies of the wild-type

protease bound to inhibitor molecules have

shown that most of the PIs occupy a similar

volume and contact similar residues within the

active site of the protease. Drug resistance

occurs where inhibitor atoms protrude beyond

the substrate envelope and contact protease

residues [76]. Thus, mutations at these sites

would specifically impact inhibitor binding while

maintaining substrate cleavage. The observation

that many of the drug-resistant mutations in the

active site do not contact the substrates has led

to the development of the substrate envelope

hypothesis: inhibitors that fit well within the

substrate envelope would be less susceptible to

drug resistance because a mutation that affects

inhibitor binding would simultaneously impact

the recognition and processing of themajority of

the substrates [76]. Of the currently prescribed

inhibitors the most potent is darunivar (DRV)

and, although not designed with the substrate

envelope constraint, DRV fits well within this

volume [2,77]. DRV in combination with ritinovir

has a high genetic barrier to resistance [78].

Developing robust HIV-1 PIs that avoid drug

resistance has proven to be a challenging task,

and the substrate envelope hypothesis provides

an approach to solving this problem. A survey of

five approved drugs using quantitativemeasures

of the bound inhibitor outside the substrate

envelope indicated that the exterior volume of

the inhibitors correlated with the loss of affinity

to mutant proteases [79]. The ability of the

substrate envelope to correlate with resistance

mutations prompted the prospective design of

new inhibitors with a lower likelihood of

developing resistance [3,80–83]. These studies

assist in validating the use of the substrate

envelope hypothesis [84] in structure-based

drug design of novel HIV PIs and have yielded

several leads for potential new drugs.

Hepatitis C NS/3A protease inhibitors

Drug resistance is also a major obstacle in the

treatment of hepatitis C virus (HCV). An esti-

mated 180 million people worldwide are

infected with HCV [85]. The essential HCV NS3/



Features

�PERSPECTIVE

4A protease is an attractive therapeutic target

responsible for cleaving at least four sites along

the viral polyprotein. Many PIs are currently in

clinical trials; however, multidrug resistance is

widespread and arises very quickly. A recent

study [86] compares the cocrystal structures of

substrate structures with cocrystal structures of

inhibitor complexes and shows that, as in the

case of HIV-1 protease [77,79,81–83,87,88], pri-

mary drug resistance occurs in HCV NS3/4A

where the inhibitors protrude away from the

substrate envelope.

These findings suggest a general model for

using the substrate envelope approach to pre-

dict patterns of drug resistance in other quickly

evolving diseases. For drug resistance to occur,

mutations must selectively weaken enzyme

affinity for an inhibitor without significantly

altering its activity. Mutations occur outside the

substrate envelope to achieve this effect,

because these molecular changes can selectively

alter inhibitor binding without compromising

enzyme activity. Whenever the interaction of a

drug target with its biological substrates can be

structurally characterized, we predict that drugs

designed to fit within the substrate envelope will

be less susceptible to resistance. Structure-based

design strategies can incorporate this as an

added constraint to develop inhibitors that fit

within the substrate envelope. As a general

paradigm, design efforts incorporating the

substrate envelope approach can lead to the

development of more-robust inhibitors that are

less susceptible to resistance.

Concluding remarks

Drug resistance negatively impacts the lives of

millions of patients and costs our society billions

of dollars by limiting the longevity of many of our

most potent drugs. Drug resistance develops so

rapidly that new drugs quickly become less

effective, as witnessed by the evolution of

resistance to existing anticancer and antimicro-

bial agents (antibiotics, antivirals, antifungals

and antiprotozoals). A recent survey of European

intensive care doctors indicated that 50% have

treated at least one patient with a Gram-nega-

tive pathogen that was totally or almost totally

resistant to all antibiotics during the previous six

months’ [89].

Drug resistance research has been very much

a disease-specific endeavor, which has limited

intellectual progress and breakthroughs. How-

ever, as shown here, we have highlighted many

parallels among quickly evolving diseases, such

as cancer and infectious disease. No single

approach to develop drugs that are less prone to

resistance will work for every drug target. Rather,


several approaches will be needed to success-

fully develop new drugs that will work on targets

where resistance is an issue. One such approach

that we have highlighted in this Feature is to

define targets that are resilient and to then

develop robust inhibitors for those targets.

Acknowledgements

The authors thank John D. Williams, PhD, and

Bing Li, PhD, of Microbiotix, for technical

assistance with this manuscript. National

Institute of Health supports the investigators as

follows: ACA – R01 AI073375, R01 GM067542;

MPP – R01 AI082577; CAS – R01 AI085051, P01

GM66524, R01 GM65347.

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Amy C. Anderson1,2

Michael P. Pollastri1,3

Celia A. Schiffer1,4,*

Norton P. Peet1,51Institute for Drug Resistance,University of Massachusetts Medical School,364 Plantation Street, Worcester,MA 01605-2324, USA2Department of Pharmaceutical Sciences,University of Connecticut, 69 N. Eagleville Road,Storrs, CT 06269, USA3Department of Chemistry & Chemical BiologyNortheastern University,417 Egan Research Center,360 Huntingdon Avenue, Boston,MA 02115, USA4Department of Biochemistry and MolecularPharmacology, University of MassachusettsMedical School, 364 Plantation Street, Worcester,MA 01605-2324, USA5Microbiotix, Inc., One Innovation Drive, Worcester,MA 01605-4306, USA


http://hivdb.stanford.edu/

http://www.who.int/vaccine_research/diseases/viral_cancers/en/index2.html

http://www.who.int/vaccine_research/diseases/viral_cancers/en/index2.html

http://dx.doi.org/10.1073/pnas.1006370107

The challenge of developing robust drugs to overcome resistance

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