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

619 ABC PROTEINS, THE FASCINATION, THE POLITICS, THE POTENTIAL FOR APPLICATIONS FOR IMPROVING HUMAN HEALTH I BARRY HOLLAND 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 excellent introductory overview and in other chapters 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 dramatically in the mid-1980s with the realization that P-glycoprotein (Pgp), responsible for multidrug resistance and a serious obstacle to effective 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 FASCINATION 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 ABC Proteins: From Bacteria to Man ISBN 0-12-352551-9 30 CHAPTER dramatically and still quite extraordinarily, despite more than 15 years now of close aquaintance 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 billion years of evolution and wide dissemination throughout all living organisms If (when) finally we get our hands on life forms from Mars and beyond we shall be very surprised if ABC proteins 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 addition, we are extremely curious to understand how the resulting release of energy is used to facilitate the action or ‘opening’ of the corresponding transport pathway and, finally, how actual movement of molecules through the membrane is accomplished Recently the ABC picture has been even further enriched, if that were possible, by the realization that there also exist more distant cousins of the ABC membrane transporters These use Copyright 2003 Elsevier Science Ltd All rights of reproduction in any form reserved 620 ABC PROTEINS: FROM BACTERIA TO MAN 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 nature of the crosstalk between ABC monomers which is required to activate and then to utilize the energy released in an ordered way THE POLITICS The stimulation and attraction of working with ABC proteins, aside from the intellectual challenge 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 morbidity and premature death This surely provides the extra incentive and motivation for the scientist to figure out how such proteins function in the ‘hope’ of effecting ‘cures’ At the same time it is in the nature of the broad canvas of academic 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 multidrug resistance in cancer chemotherapy, respectively This is a fine, highly motivated sentiment and clearly in this case containing an element of truth However, such sentiments 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 hospitals and pharmacies In reality, in the real laboratory world of research directors, students and postdocs, fundamental research at the frontier is slow and painstaking, progress incremental, requiring infinite patience and ingenuity to test and discard many hypotheses before making real groundbreaking discoveries Research is also about training oneself to think constructively and creatively and, above all for the experienced 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 exploitation of basic knowledge of ABC proteins, a final comment on the realities facing current scientists 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 fundamentals 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 DIFFICULTIES, REALITIES AND PROSPECTS FOR APPLICATIONS Successful application or exploitation of knowledge gained from academic studies is not a simple 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 ABC PROTEINS APPLICATIONS FOR IMPROVING HUMAN HEALTH 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 tackling 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 interconnected masses of metabolic and intercellular circuits, with gene expression differentially regulated in different tissues and phases of life, operating at the optimum balance evolved over many millions of years Modifying or delivering replacements for defective ABC genes which function perfectly in situ, or designing medicines which precisely counter the expulsion 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 screening for CFTR alleles in the population over the last decade In fact the application of fundamental knowledge in the area of susceptibility testing 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 proteins, 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 technology 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 paradoxical fact that drugs like glibenclamide, used each day by millions of patients suffering from type diabetes in order to stimulate insulin secretion, were identified and developed through empirical techniques many years before its target protein, SUR, was discovered and characterized Nevertheless, we might anticipate for the future, still some way off, that from highresolution structures of SUR, combined with better understanding of its molecular functioning and its precise contribution in the physiological 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 specific target compared with the highly conserved ABC domain Here perhaps is a constructive lesson for elaborating designer drugs effective against the highly specific transport domain of other ABC proteins, such as the multidrug transporters 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 microorganisms 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 clinical 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 Moreover, 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 621 622 ABC PROTEINS: FROM BACTERIA TO MAN include ABC transporter assays in early screens in drug development programs, in order to eliminate drugs that are transported by these drug pumps PREVENTION AND TREATMENTS OF DISEASE: THE MOLECULAR FUTURE 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 treatments This will be based on fundamental knowledge of how the cells, tissues and organs of the human body actually work at the molecular level This is manifestfully not because the empirically based procedures are not effective but that armed with informed insight we can hope ultimately to 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 laboratories, showing that this mutant protein folds incorrectly Much to our frustration, however, our inability to understand why this mutant misfolds 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 administering 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 procedures 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 PERSPECTIVES Thus, whilst an effective treatment for cystic fibrosis is yet some way off, we must not forget that determination and optimism are the essential 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, membrane proteins, is now embracing and benefiting from the new frontier of biology, the exploitation of biophysical and sophisticated spectroscopic techniques, to yield vital highresolution 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 proteins will require not only global understanding of their physiological role but also the molecular and atomic level detail required to understand the dynamics of how these proteins and their associates maneuver and change conformation as they function Much more research is required to meet these demands, ideally with enlightened funding regimes These should provide for and inspire ‘riskier’ ABC PROTEINS APPLICATIONS FOR IMPROVING HUMAN HEALTH creative thinking in basic research in the public sector, at least in wealthy states, unfettered by pressures to relevant research Support for such ‘blue skies’ research should, however, also include provision for better opportunities, when and where appropriate, for academics to collaborate 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 underdeveloped 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 concluding chapter for myself, on behalf of all the editors, to thank most warmly all the participants involved in the preparation of this volume; equally we acknowledge and applaud the efforts of the many others, past and present in the laboratories of the book’s major contributors, who in the end make all our achievements both possible and enjoyable; and of course we are heavily indebted to the ever larger community 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 inspiring account of many if not all of these fascinating and important ABC proteins 623 ... number of such potential applications in relation to the ABC field are already in view For ABC PROTEINS APPLICATIONS FOR IMPROVING HUMAN HEALTH example, we can certainly anticipate for the near future... 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... proteins: the mechanism of intra-molecular signaling between the ABC and the membrane domain (or DNA), and the nature of the crosstalk between ABC monomers which is required to activate and then to