CHAPTER 30 – ABC PROTEINS, THE FASCINATION, THE POLITICS, THE POTENTIAL FOR APPLICATIONS FOR IMPROVING HUMAN HEALTH

CHAPTER 30 – ABC PROTEINS, THE FASCINATION, THE POLITICS, THE POTENTIAL FOR APPLICATIONS FOR IMPROVING HUMAN HEALTH CHAPTER 30 – ABC PROTEINS, THE FASCINATION, THE POLITICS, THE POTENTIAL FOR APPLICATIONS FOR IMPROVING HUMAN HEALTH CHAPTER 30 – ABC PROTEINS, THE FASCINATION, THE POLITICS, THE POTENTIAL FOR APPLICATIONS FOR IMPROVING HUMAN HEALTH CHAPTER 30 – ABC PROTEINS, THE FASCINATION, THE POLITICS, THE POTENTIAL FOR APPLICATIONS FOR IMPROVING HUMAN HEALTH

This chapter is dedicated to the memory of a

remarkable young scientist, Julian Boucher, a

truly inspiring colleague, who died in 1999.

Studies of ABC proteins, in the form of HisP

and MalK, were already well under way in the

early 1980s and these are described in the

excel-lent introductory overview and in other

chap-ters of this volume The select band of devotees

involved in these initial studies were fascinated

by the mechanism of histidine and maltose

uptake in Gram-negative bacteria, certainly an

esoteric subject Everything changed

dramati-cally in the mid-1980s with the realization that

P-glycoprotein (Pgp), responsible for

multi-drug resistance and a serious obstacle to

effec-tive antitumor chemotherapy, was also an ABC

transporter This was followed quickly by the

identification of the CFTR protein as a novel

ABC transporter, and the subject has never

looked back, with now thousands of ABC genes

in the database and the avalanche continues as

new genome sequences accumulate

The reason for the fascination of ABC proteins

and their associated partners, however, does not

stop at the sheer size of this superfamily but is

compelling, as this volume so demonstrably

illustrates, because of the enormous breadth of

biological processes that they embrace More

dramatically and still quite extraordinarily, despite more than 15 years now of close aquain-tance with these proteins, remains the fact that these processes are driven by essentially the same ubiquitous ATPase This is a molecular machine still easily recognizable by virtue of sequence motifs, sufficently unchanged as to be detectable by ‘eye’ despite more than three bil-lion years of evolution and wide dissemination throughout all living organisms If (when) finally

we do get our hands on life forms from Mars and beyond we shall be very surprised if ABC pro-teins and ABC transporters are not represented

The attraction and indeed beauty of ABC transporters is that their study unites on the one hand many varied disciplines, and on the other, more importantly, brings together scientists with interests in quite disparate organisms found in all conceivable niches on the planet All

of us are engaged in the hunt for the common principles that govern the mechanism whereby

so many different molecules or ions trigger the different ATPase machines into action In addi-tion, we are extremely curious to understand how the resulting release of energy is used to facilitate the action or ‘opening’ of the corre-sponding transport pathway and, finally, how actual movement of molecules through the membrane is accomplished

Recently the ABC picture has been even fur-ther enriched, if that were possible, by the reali-zation that there also exist more distant cousins

of the ABC membrane transporters These use

ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9

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30

F ASCINATION , THE P OLITICS ,

THE P OTENTIAL FOR

A PPLICATIONS FOR I MPROVING

H UMAN H EALTH

CHAPTER

ATP to effect some critical steps in polypeptide

synthesis, DNA repair or recombination It is

not yet clear to the transporter aficionados how

to reconcile the common principles of action of

such ABCs with those involved in import or

export Nevertheless, these principles are surely

there, involving perhaps the most fascinating

secrets of these proteins: the mechanism of

intra-molecular signaling between the ABC and

the membrane domain (or DNA), and the

nat-ure of the crosstalk between ABC monomers

which is required to activate and then to utilize

the energy released in an ordered way

The stimulation and attraction of working with

ABC proteins, aside from the intellectual

chal-lenge of simply knowing how they work, is

undoubtedly for many of us that some of these

proteins are ‘useful’ in relation to the human

condition In some cases when the human ABC

machine malfunctions, sadly this can bring

mor-bidity and premature death This surely

pro-vides the extra incentive and motivation for the

scientist to figure out how such proteins

func-tion in the ‘hope’ of effecting ‘cures’ At the same

time it is in the nature of the broad canvas of

aca-demic research that scientists instinctively study

all manner of topics, relevant or completely

irrelevant (for the moment), simply because

there are always new truths to be discovered

everywhere Certainly, however, studying an

ABC protein with the most trivial of roles in the

most obscure of organisms can be perceived as

justified because it belongs to the superfamily

that contains CFTR, Pgp and MRP, and hence

the opportunity to contribute to curing cystic

fibrosis, or the alleviation of problems of

mul-tidrug resistance in cancer chemotherapy,

respectively This is a fine, highly motivated

sen-timent and clearly in this case containing an

ele-ment of truth However, such sentiele-ments are

easily colored by unreal expectations, and an

understandable degree of self-delusion, shared

by scientist and public alike, in relation to what

practical dividends may actually stem from

basic research

Increasingly, therefore, we are asked to justify our research in terms of the resulting benefits to

society, leading us to succumb too frequently,

although with the highest of intentions, to the

employment of certain artifices to meet the

demands of funding agencies Unfortunately, this

in turn leads to some unwelcome repercussions,

with the perception of science suffering when

we fail to deliver new products and therapies rapidly from the laboratory bench into the hos-pitals and pharmacies In reality, in the real lab-oratory world of research directors, students and postdocs, fundamental research at the fron-tier is slow and painstaking, progress incremen-tal, requiring infinite patience and ingenuity to test and discard many hypotheses before mak-ing real groundbreakmak-ing discoveries Research is also about training oneself to think construc-tively and creaconstruc-tively and, above all for the expe-rienced scientist, to inspire and guide the next generations to think creatively, to critically weigh evidence, and to formulate conclusions based

on informed judgments Happily, ABC protein research is a rich and fertile field in which to express and learn such skills

Before moving on to the topic of the exploita-tion of basic knowledge of ABC proteins, a final comment on the realities facing current scien-tists Academics, like our corporate colleagues, are increasingly subject to the same pressures to

‘perform productively’, to publish to fill quotas rather than to prove theories Not surprisingly, this increases the tendency towards research without risk, publishable but non-contentious research that skims the initial descriptive cream

of a new phenomenon or an old phenomenon in

a new organism, before moving on to repeat the same formula Digging deep into the fundamen-tals of a subject, where the going becomes slow, tough and above all risky, is not at all attractive All these comments apply in the ABC field as to any other, and in surveying the mass of recent publications, for example, on prokaryote ABC proteins, it is clear that the overwhelming majority are simply describing new examples;

we encourage more to wrestle with the basic principles, despite the obstacles

Successful application or exploitation of knowl-edge gained from academic studies is not a sim-ple matter, and like basic research also takes time, patience and flair, perhaps also an element

of luck and certainly should also include exhaustive attention to detail Bearing this in mind a number of such potential applications in relation to the ABC field are already in view For

example, we can certainly anticipate for the

near future that many of us could be diagnosed

as having an ABC protein not quite optimum

for a long life of perfect control of cholesterol

levels, a situation which could respond perhaps

to some future molecular tweaking to relieve

the pressure on our arteries Whilst in principle

we can already envisage, with regard to

tack-ling such problems of human health at the

genetic level, the exploitation of fundamental

knowledge to achieve the necessary genetic

engineering and gene therapy, actually carrying

this into practice is far from trivial All higher

organisms are incredibly complex

intercon-nected masses of metabolic and intercellular

circuits, with gene expression differentially

reg-ulated in different tissues and phases of life,

operating at the optimum balance evolved over

many millions of years Modifying or

deliver-ing replacements for defective ABC genes

which function perfectly in situ, or designing

medicines which precisely counter the

expul-sion of antitumor drugs, without disrupting

other physiological functions of Pgp, MRPs or

the other ABC proteins, is a tall order

Nevertheless, these are feasible and laudable

objectives, which will require comprehensive,

dedicated research in model microorganisms,

in animals and finally in rigorous clinical trials

in humans in order to fulfill them Even then

we cannot evade the reality that success cannot

be guaranteed no matter how smart we are We,

our peer reviewers and our support providers,

have to learn (or relearn) to accept therefore the

concept of sometimes failing in such endeavors;

equally importantly to accept the concept of

starting over with a new strategy when needed,

no matter how expensive or inglorious

Notwithstanding the difficulties, exploitation

of knowledge from fundamental and applied

studies of ABC proteins should ultimately bring

some long-term returns Important benefits in

diagnostics have already accrued in the

screen-ing for CFTR alleles in the population over the

last decade In fact the application of

fundamen-tal knowledge in the area of susceptibility

test-ing and diagnostics will probably continue to

lead the way in the discovery of new treatments

for disease for some time to come We certainly

may anticipate greatly increased use of gene

diagnostic probes for screening for potentially

disadvantageous alleles of several ABC

pro-teins, including the ABCA1 protein involved in

cholesterol trafficking

Treatments of human diseases arising directly from the results of academic or fundamental

research and concomitant advances in technol-ogy are still very much in their infancy Thus, they lag far behind treatments arising primarily from purely empirical discovery of drugs and procedures A good example of this is the para-doxical fact that drugs like glibenclamide, used each day by millions of patients suffering from type 2 diabetes in order to stimulate insulin secretion, were identified and developed through empirical techniques many years before its target protein, SUR, was discovered and charac-terized Nevertheless, we might anticipate for the future, still some way off, that from high-resolution structures of SUR, combined with better understanding of its molecular function-ing and its precise contribution in the physiolog-ical context of insulin regulation, it may be

possible to design drugs which slot precisely into

a specific pocket of the target structure with minimal side effects Interestingly, in the case of glibenclamide, the site of action has been traced

to the membrane domain of SUR, a region of the molecule likely to constitute a much more spe-cific target compared with the highly conserved ABC domain Here perhaps is a constructive les-son for elaborating designer drugs effective against the highly specific transport domain of other ABC proteins, such as the multidrug trans-porters in humans, pathogenic microorganisms and parasites, rather than the ABC ATPase

There is clearly great interest now in developing drugs against such transporters, which on the one hand can limit the effectiveness of cancer chemotherapy, or, increasingly, pose a serious threat in the form of multidrug resistant micro-organisms or other pathogens, on the other At the present time we are limited to screening for such drugs by empirical procedures in the absence of the atomic level structures of the transporter Such structures are an absolute requirement for future rational drug design

For the moment no effective drugs against such ABC proteins have yet made it into clini-cal practice In contrast, increasingly, human multidrug ABC transporters are being put to good use either as dominant selective markers for concomitant transfer of a ‘corrective’ gene

in relation to gene therapy, or through transient expression in transfected bone marrow cells in order to provide protection against cytotoxic anticancer drugs during chemotherapy More-over, in recognition that Pgps and the MRP-type drug transporters are likely to play significant roles in the absorption, tissue distribution and elimination of many new potential drugs (see Chapter 18), pharmaceutical companies now

include ABC transporter assays in early screens

in drug development programs, in order to

eliminate drugs that are transported by these

drug pumps

A major objective for the next twenty years in

regard to human health care is of course to shift

the balance decisively away from empirically

based treatments and drug discovery, towards

informed procedures for prevention and

treat-ments This will be based on fundamental

knowledge of how the cells, tissues and organs

of the human body actually work at the

molecu-lar level This is manifestfully not because the

empirically based procedures are not effective

but that armed with informed insight we can

hope ultimately to do far better Conventional

therapies have resulted in substantial increases

in life expectancies for cystic fibrosis sufferers

but still the disease takes away from us young

persons with lives unfulfilled No doubt more

developments in conventional methods will

give more progress yet, particularly perhaps in

countering bacterial infections of the airways in

CF patients However, much hope is pinned on

gene therapy or treatments to specifically rescue

the function of the major mutant protein in the

Caucasian population, the deletion F508 In the

latter case, the approach to novel treatments

stems directly from basic studies of the CFTR

protein and its gene in many academic

laborato-ries, showing that this mutant protein folds

incorrectly Much to our frustration, however,

our inability to understand why this mutant

mis-folds severely hampers our attempts to design a

cure We shall understand such riddles in the

medium future but for the moment we must

rely on less precise procedures, by

administer-ing empirically derived compounds which may

bind the mutant CFTR and suppress the folding

defect Unfortunately, few ligands with high

affinity for CFTR are so far available Much

effort has been put into the even more ambitious

quest for a gene therapy for CF patients over

the last decade and is still ongoing Various

approaches have been tried, including gene

delivery into the airways by disarmed viral

vectors or transfection by either naked DNA or DNA packaged with cationic liposomes The feasibility of at least transient expression of the CFTR protein in respiratory epithelial cells from DNA administered by relatively simple proce-dures has been demonstrated and at least 20 clinical trials worldwide had already been reported by 1997 From these and subsequent trials it appears that these procedures are safe but so far not effective clinically

Thus, whilst an effective treatment for cystic fibrosis is yet some way off, we must not forget that determination and optimism are the essen-tial characteristics for most successful scientists Similarly, as editors of this, we hope, landmark volume, celebrating the joys and excitement of prizing from nature the secrets of ABC systems,

we take pride and hope in looking to the future for further major advances in fundamental knowledge Encouragingly, the study of this ABC superfamily, albeit in most cases including the most refractory of macromolecules, mem-brane proteins, is now embracing and benefit-ing from the new frontier of biology, the exploitation of biophysical and sophisticated spectroscopic techniques, to yield vital high-resolution structural information This is already showing dividends with crystal structures of several ABC domains very recently solved and now, the first of many, we hope, the structure of

an entire molecule, MsbA This we equally hope will be the prelude to the application of even more novel techniques which would reveal the dynamic properties of ABC proteins

as they move their transport substrates through membranes and along polypeptide chains or DNA molecules

Notwithstanding the need for patience and rigor in research (tempered by more realistic expectations) any successful applications designed to alleviate suffering and to enhance the human condition in relation to ABC pro-teins will require not only global understanding

of their physiological role but also the molecu-lar and atomic level detail required to under-stand the dynamics of how these proteins and their associates maneuver and change con-formation as they function Much more research is required to meet these demands, ideally with enlightened funding regimes These should provide for and inspire ‘riskier’

creative thinking in basic research in the public

sector, at least in wealthy states, unfettered by

pressures to do relevant research Support for

such ‘blue skies’ research should, however, also

include provision for better opportunities,

when and where appropriate, for academics to

col-laborate in a whole variety of ways with the

corporate sector in advancing the exploitation

of their basic research This is common practice

in the United States, but is woefully

under-developed in Europe Finally, how we as a global

society identify urgent problems of human

health for priority attention and then how to

mobilize our resources worldwide in the best

way to meet the challenge are also in need of

radical review, but that would be outside the

scope of this text

It only remains now at the end of this con-cluding chapter for myself, on behalf of all the editors, to thank most warmly all the partici-pants involved in the preparation of this vol-ume; equally we acknowledge and applaud the efforts of the many others, past and present in the laboratories of the book’s major contribu-tors, who in the end make all our achievements both possible and enjoyable; and of course we are heavily indebted to the ever larger commu-nity of scientists worldwide working on ABC proteins, who, whilst not having contributed directly to this volume, have provided a vast store of published work that we have plundered

in the hope of producing a balanced and inspir-ing account of many if not all of these fascinat-ing and important ABC proteins