Pharmaceutical biotechnology Editorial overview

Frank McCormick and Wendell Wierenga

Onyx Pharmaceuticals, Richmond and Parke-Davis, Ann Arbor, USA

Current Opinion in Biotechnology 1995, 6:621-623

Following its birth in the 1970s, biotechnology was embraced by nearly every pharmaceutical research institution in anticipation of its impact on drug discovery. In addition, the birth ofbiotechnology led to the genesis of many companies as entrepreneurial research ventures, which sought to exploit the new technologies as a source of new and potent treatment therapies. Although the 1980s witnessed this broad embrace, the true scope and significance ofbiotechnology in pharmaceuticals was, at that time, hard to quantify. Would the impact be limited to proteins as drugs? Would monoclonal antibodies revolutionize therapeutics? Would biotechnology impact genetics? Would the typical small molecular weight organic molecule continue to be a source of novel drugs for the future and, if so, how would biotechnology alter this traditional drug discovery process?

The current decade has revealed the answers to these and many other questions, and we can now quantify much more adequately the impact of biotechnology on drug discovery. This issue of Current Opinion in Biotechnology seeks to describe the breadth and the depth of that impact. The following reviews provide the reader with a thorough understanding of the future of proteins as drugs, the use of biotechnology for new macromolecular targets, the impact on drug discovery through combinatorial chemistry and high-throughput screening, structure-based drug design, novel delivery systems, and gene therapy.

Biotechnology has provided a multitude of novel molec- ular targets that can be utilized in assays for screening potential drugs from libraries of molecules. This process will be further fueled by genomic sequencing efforts and high-throughput cDNA sequencing that continues to generate new potential targets. Biotechnology has also enabled the design, in vitro, of functional systems that mimic signaling pathways in vivo, yielding cellular pharmacology relevant to normal as well as pathological disease states. This, in turn, has spawned an interest in libraries of molecules and combinatorial chemistry to generate these libraries. As defined b y Gordon (pp 624-631) in his article on libraries of non-polymeric organic molecules, combinatorial chemistry is the 'systemic and repetitive covalent connection of a set

of building blocks of varying structures to each other to yield a large array of molecular entities'. This new paradigm proceeded from the initial work creating polymeric libraries of peptides and oligonucleotides, which also can be screened as potential sources of novel drugs. The current emphasis is on organic molecules and the creation of novel chemistries, coupled with automation and creative analytical chemistry, to enable the rapid synthesis and analysis of an array of molecules representing a particular structural database. Gordon outlines the general approaches, using as an example peptidylphosphonate libraries, which are transition state-based inhibitors of metalloproteinases. In addition, he describes mercaptoacyl proline libraries as angiotensin-converting enzyme inhibitors, peptide-turn mimetic libraries, and various heterocyclic arrays.

A key element in exploiting combinatorial chemistry in large libraries is the tracking and identification of the synthesized compounds, particularly those that are active in a subsequent screening assay. Chabala (pp 632-639) describes encoded combinatorial chemistry using the solid phase as a key strategy for accomplishing this purpose. Initially, approaches such as 'split synthesis' and deconvolution strategies were utilized. Chemical tagging, however, is clearly versatile and a significant advance over earlier methods. Chabala details tagging procedures using sequenceable biopolymers and then describes novel methods using electrophoric organic molecules as part of a binary code. This approach is illustrated by discussing binary encoded libraries of acylpiperidines and dihydrobenzopyrans, which have yielded nanomolar-level inhibitors of carbonic anhy- drase. These libraries comprised 1000-6000 members.

DeWitt and Czarnik (pp 640--645) describe another concept in automated rapid synthesis of low molecular weight organic molecules. The outlined goal is one of securing a small library for a second phase of expanding the structure-activity relationship profile of an already identified lead. This technique has been termed Diversomers and, in essence, involves a parallel processing architecture involving automated solid-phase synthesis. It has been applied to the synthesis of quinolones, benzadiazapines, and hydantoins. The thrust

Abbreviations IL--interleukin; VEGF---vascular endothelial growth factor.

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of all this is that rapid high-volume screening and library synthesis through combinatorial chemistry methods will be an integral part of drug-discovery strategies in any pharmaceutical R&D environment.

Although it made is debut in the 1980s, computer-aided drug design has, nonetheless, begun to significantly impact drug discovery only in the current decade. The obligatory role ofbiotechnology in structure-based drug design was to provide the appropriate proteins, be they receptors, enzymes, antibodies or the hke, in sufficient quantity and purity to support structure-elucidation techniques such as NM1K spectroscopy and X-ray crystallography. This combination of computer-aided drug design with structure-based approaches is updated in the article by Jackson (pp 646-651). Included are brief overviews of various structure-based drug discovery projects such as HIV protease, matrix metalloproteinases, interleukin-l~ converting enzyme, purine nucleoside phosphorylase, elastase and several polymerases. In addition, techniques such as three-dimensional quan- titative structure-activity relationship (QSAR) analysis, comparative molecular field analysis, and homology modehng are described. These involve components of computer-aided drug design, but utilize elements other than the direct structure to predict potential structures that interact with the active sites of the molecular targets.

Indeed, the integration of structural data with modehng techniques has yielded a procedure called 'docking', which is described by Jones and Willett (pp 652-656). This involves the prediction of structure and binding free energies with a hgand-receptor complex on the basis of only the structures of the free hgand and the receptor. Docking represents an algorithm with various degrees of flexibility in fitting hypothetical ligands into low-energy conformations of the requisite macromolecule. The challenge, of course, is the significant number of variables involved, as well as the need to search a large database of structures. Nonetheless, this is a powerful technique with growing importance in 'screening' electronic files of chemical databases. This virtual screening technique has led to various novel unanticipated leads that have formed the basis of lead optimization in drug-discovery approaches.

Much of the focus for biotechnology and its applications to drug discovery has been on extracellular enzymes and receptors. As intracellular signahng pathways have been elucidated over the past few years, it has become apparent that these effectors of signahng pathways, be they enzymes, binding proteins or lower molecular weight ligands, could also be targets for drug discovery. Protein phosphorylation has been an intriguing area of research because it represents an important regulatory mechanism for protein activation/deactivation. In fact, several kinases have been identified that phosphorylate various amino acids, such as serine, threonine, tyrosine or histidine, in either a trans mode or an autophos- phorylation mode, which usually is an obhgatory

component for activation and continuation of a signaling mechanism. Lee and Adams (pp 657-661) describe the cytoplasmic serine/threonine kinases as important effectors in signal- ing pathways. These ATP-dependent kinases are suscep- tible to several different types of inhibitor: catalytic site inhibitors, regulatory site inhibitors, and substrate-based inhibitors. Early work studied the inhibition of protein kinase C by several hetemcyclic-based molecules, some of which were natural products. More recently, the focus has been on kinases such as CSBP, mitogen-activated protein kinase, protein kinase A and the cyclin kinases. Lee and Adams detail examples of inhibitors and their use in cellular pharmacology. The search for catalytic site kinase inhibitors was associated with the dogma that all such inhibitors would be non-specific because all kinases require ATP in the catalytic site. Yet, recent work has revealed some remarkably potent (picomolar level) inhibitors of tyrosine kinases, which are ATP dependent and have remarkable selectivity. Fry and Bridges (pp 662-667) describe examples of inhibitors of epidermal growth factor and platelet-derived growth factor receptor tyro- sine kinases that are extremely potent and remarkably specific. Work continues at a rapid pace in this area, including the search for inhibitors of cytosolic tyrosine kinases such as c-Src and related family members. Several of these inhibitors have demonstrated antiproliferative activity in vivo, and it is anticipated that this research, together with the search for inhibitors serine/threonine kinases, will yield interesting and specific therapeutic strategies for the treatment of cancer in the future. As signal transduction pathways continue to be unrav- eled, new opportunities for therapeutic intervention are revealed. Symons (pp 668-674) describes pathways activated by Ikas oncoproteins in transformed cells: these pathways act synergistically to give the full transforming effect of the Ras protein. This synergy imphes that inhibition of one of these pathways leads to effective inhibition of the other, and indeed this has been demonstrated genetically. Hopefully, small-molecule drugs that target components of these pathways will form the basis of future cancer treatments. Furthermore, the strong synergistic effects seen in multiple pathways activated in cancer cells may not apply to normal cells, in which these pathways are seldom activated concurrently. A clear understanding of these issues might provide selectivity for therapeutics in the future, when the full consequences of these interactions is appreciated. Likewise, Martiny-Baron and Marm~ (pp 675-680) demonstrate how a clear understanding of a specific growth factor, vascular endothelial growth factor (VEGF), in the complex biological process of vascularization and tumor progression, leads to a number of therapeutic opportunities. Having established the importance of the VEGF pathway, specific proteins can be targeted for inhibition. This could involve blocking ligand binding to the receptor, inhibition of receptor activation (the receptor is another kinase), or blocking

Editorial overview McCormick and Wierenga 623

critical downstream pathways. Clearly, strategies targeted at the receptor itself would have most specificity.

The application of biotechnology to generate recom- binant protein targets for drug discovery continues to be the focus for research. The first and, so far, most successful products of the industry have been protein drugs. Koths (pp 681-687) reviews the most important issues related to protein drug development, whether for recombinant vaccines or for therapeutics. As a wealth of knowledge on protein drug development has accu- mulated, many of these issues, such as immunogenicity, biodistribution, stability, and protein folding, have been examined and, in a large number of cases, resolved empirically over the past decade. An example of the clinical development of one protein drug is provided by Alam (pp 688-691), who focuses on interferon-~-lb, a molecule that is currently approved for the treatment of multiple sclerosis, but has potential in many other areas. Hunt and Foote (pp 692-697) then describe the discovery and development of a novel human cytokine with tremendous clinical potential: thrombopoietin or Mpl ligand. This drug is at a relatively early stage of development, but the technical issues facing its further refinement are undoubtedly related to those faced and dealt with over the previous decade. Clearly, the number of therapeutically useful protein drugs has a limit, but this recent example suggests many more remain to be discovered.

The advent of protein drugs, not unexpectedly, has brought new challenges in drug delivery. It is recognized, however, that the drug delivery problem is not unique to large molecular weight entities such as proteins. Indeed, over the past 30 years, research has been focused on methods for enhancing drug bioavailabil- ity, reducing toxicities, targeting specific organs and modifying drug pharmacokinetics. Liposomes have been one of the major areas for research in drug delivery. Chonn and Cullis (pp 698-708) outline the history of liposome research and bring us up to date with new advances, including fusogenic liposomal systems. Important challenges in liposome research include enhancing circulation time of the drug, maximizing drug loading and optimizing target delivery. The last of these has been a significant challenge. Most of the approaches for targeted delivery have involved antibody-mediating

targeting. Although this has achieved only limited success, to date, low molecular weight drugs, such as amphotericin and doxorubicin, have successfully negotiated clinical evaluation to reach the marketplace in liposomal formulations. Significant research is currently under way, including clinical evaluation of various proteins, such as interleukin (IL)-2 and IL-7, in lipsomal formulations. A significant challenge for the delivery of highly charged molecules, such as oligonucleotides, is intracellular delivery. The use of cationic lipids and their variations in liposomal formulations is a growing research area because efficient intracellular uptake is prerequisite to the delivery of antisense, ribozymes, and genes. Limited success has been achieved, but there is still significant room for improvement. All the indications are that novel non-toxic liposomal formulations will have successful applications in delivering these unique highly specific oligomeric anions into cells and tissues.

A new and promising application of biotechnology to human therapeutics is gene therapy. Cunliffe, Thatcher and Craig (pp 709-713) describe some innovative aspects of this technology, which is still in its infancy. The first concept ofgene therapy involved the stable expression of a gene in a cell normally lacking this gene function, so that a wild-type phenotype is restored. More recently, gene therapy has been discussed in the context of gene delivery to cancer cells as a means of directing their destruction. In either case, considerable challenges remain: delivery problems, instability of expression, immune responses to treated cells, et cetera. To some degree, these are similar to the problems that faced the pioneers of biotechnology in the late 1970s and 1980s, when the production of recombinant proteins at commercial scales, and the potential problems with formulation and antibody responses, seemed formidable. It will be of great interest to see whether the innovators of gene therapy are as successful as their predecessors in surmounting the problems at hand.

F McCormick, Onyx Pharmaceuticals, 3031 Research Drive, Building A, Richmond, California 94806, USA. W Wierenga, Parke-Davis Pharmaceutical Division, Warner- Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105-2430, USA.