Structure-based drug design: applications in immunopharmacology and immunosuppression

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Manuel A. Navia and Debra A. Peattie Structure-based drug design W3DD) combines the power of many scientific disciplines, such as X-ray crystallography, nuclear magnetic resonance, medicinal chemists, molecular modeling, biology, enzymology and biochemistry, in a functional paradigm of drug development. The current StYengfh of SBDD lies in parlaying enzyme inhibitors into drugs, but a variety of technological advances over the past few years now makes it possible to address complex biological targets, such as those regulating immunosuppression and immunoactivation. Manuel Navia and Debra Peattie discuss the SBDD paradigm and consider several of its achjevements and challenges in immunopha~acology, purfic~larly as fhese apply to the design of novel, potent immunosuppressants. Structure-based drug design (SBDD) can be defined as the use of atomic-level three-dimensional structure info~ation about a macromolecular target to guide and assist in the design of therapeutic agents. At its current stage of evolution the SBDD approach is incapable of producing such agents without assistance from other dug-discover disciplines. A drug development campaign centered on SBDD is likely to consist of a series of tactical extrapolations from experimentallydetermined structural information, leading to the synthesis of intermediate lead compounds whose predicted properties can be subjected to experimental verification (for reviews see Refs 1 and 2 and references therein). Operationally, SBDD investigations have focused strongly on the design of enzyme ir$Gbitors2 in preference to other areas such as receptor antagonists or agonists. Under ideal circumstances, the structure of an enzyme or enzyme-inhibitor complex can define all the molecular interactions relevant to the design of potent in vitro inhibitors, where such an inhibitor need only antagonize the catalytic activity of the enzyme

M. A. Nauia is Vice President and Senior Scienfist and D. A. Peattie is Senior Scientist at Vertex Pharmacrtrdicals Incorporated, 40 Allsforr Street, Cambridge, MA 02139-4211, USA.

- much like a piece of gum in a fine watch. This is a much less formidable task than that required of a receptor agonist, for example, where the gum would have to both bind to the watch and make it run correctly. In this review, we consider the key elements of the SBDD process in the context of improving immunosuppressive drugs. Initiating the design process The SBDD paradigm is summarized schematically in Fig. 1. It consists of an initial phase in which the necessary technical components of the process are marshalled, and a production phase in which those components are applied in cyclic fashion. As part of the initial phase, the desired macromolecular ‘drug target’ must first be identified biochemically with a high degree of certainty. This is critical, since the success of the entire program will hinge on the therapeutic relevance of that selection. In practice, most of the macromolecular targets that have been examined through structure-based drug design have been proteins. SBDD is equally valid for any macromolecular target whose structure can be determined, including nucleic acids and protein-nucleic acid complexes. Subsequently, the macromolecular target must be produced and isolated in sufficient quantity and purity to allow for its crystallization and structure deter-

mination by X-ray diffraction methods. For nuclear magnetic resonance (NMR) studies of drugtarget interactions in solution, isotope labelling of protein or drug may be necessary in order to obtain structural information of sufficient accuracy to be useful. Suitable assay systems must also be defined at the initial stage, in order to measure accurately the ‘success’ of a given design initiative. As with the initial identification of the drug target, these assays must be chosen with care to mirror efficacy accurately in the disease process since much of the guidance for the design process comes from these indicators of activity. Assays will typically include determination of in vitro efficacy at the molecular level (e.g. enzyme inhibition assays), in vitro biological activity (e.g. performance in cell-culture systems), ex vivo effects in intact organs (e.g. biliary excretion in liver perfusion and systems) whole-organism studies in vivo (e.g. efficacy, pharmacokinetics, toxicity). Finally, it is desirable to identify early in the initial stage a prototype chemical lead (or ‘proto-lead’) to structurally delineate the active site or binding region of the target macromolecule and to locate a subset of critical interactions therein. In practice, this is not as unreasonable as it might seem since the therapeutic relevance of target macromolecules often stems from such a proto-lead interaction in the first place (e.g. peptidyl snake venom proto-leads were critical in develo$ng angiotensin-converting enzvme inhibitors3L In some circtmstances, a proto-lead can be designed by modification of a known natural effector or substrate [e.g. peptidyl inhibitors of renin* and human immunodeficiency virus- (HIV-) 1 proteases* were first proposed from a knowledge of the structure of substrates of those enzymes]. At times, an existing drug can serve as a protolead if its properties can be significantly improved. Topical carbonic anhydrase inhibitors, for example, might substantially improve the treatment of ocular hypertension and glaucoma’ in comparison to existing systemic agents. Even in situations where suitable proto-leads cannot be identified, computational modelbuilding procedures should be

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Fig. 1. Schematic representation of the structure-based drug design process. An initial phase is followed by cycles of structure determination by crystallography or nuclear magnetic resonance.molecularmodeling, synthetic chemistry and experimenta!, biologica! and biochemical verificz?iion.

able to jump-start the SBDD process via de nouo design of proioleads based o’n the structure of the target molecule’s surface8c9. Executionof the design process Once the SBDD process has been initiated, one enters the production phase of the paradigm, which functions in repeating cycles as shown in Fig. 1. In one version of SBDD, the structure of the current lead compound, in complex with its macromolecular target, is first solved by X-ray or NMR techniques. That structure {and the aggregate of all previous structures solved) is then examined by systematic molecular modeling methods and considered in ii&t of past experience to arrive at a series of incremental lead designs. These designs are evaluated in direct consultation with the medicinal chemists charged with executing their synthesis so that practical issues can be considered at the earliest possible point. Such practical concerns might include ease of synthesis, process sca~abi~i~, formulation

and known or suspected problems with certain classes of substituents with respect to toxicity, bioavailability and lability. Once synthesized, compounds are assayed to ascertain whether the expected theoretical improvements were realized. Unexpected results are subject to immediate structure &termination by X-ray or NMR methods and are quickly reexamined to determine which of the operating assumptions about the interaction with the drug target molecule require adjustment. If a predicted compound works as expected, one can proceed directly tc further model building and synthesis without necessarily confirming the prediction by a structure dete~ination. Even in those situations, however, it is still prudent to check periodically by experiment the validity of the assumptions underiying predictions about drug candidates since compounds have been known to behave quite satisfactorily in an assay for a reason different from that expected. Throughout the SBDD process, biochemical and

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biological investigations continue, in order to understand further the role of the target macromolecule in the disease process, to improve the quality, purity and quantity of the target macromolecule made available to the SBDD process, and to improve the reliabili~ and efficacy of assay procedures used. Role of technology Several advances in recent years have had an important effect on the SBDD paradigm. In medicinal chemistry, for example, the development of efficient regio- and stereo-selective reactions has been crucial, as have developments in sensitive, rapid and automated instrumentation to characterize the consequences of a given synthetic strategy. Similarly, new computational methods and the dramatic increase in raw computational power available per dollar have greatly helped the molecular modeling effort, enabling the routine exercise of such previously prohibitive methods as molecular dynamics, ~b it~itioc&ulations and the implementation of large-scale conformational searches in the de nouo design of drug candidate molecules. Perhaps the most remarkable technological advance is the evolution of X-ray crystallography and NMR to the status of applied sciences. In C~stalIograpby, this has been made possible by three main improvements in instrumentation: area detector technology for the rapid collection of X-ray data which can now be completed in days versus months or even years with the early fi!m methods (see Fig. 2); high-performance workstations that can execute general crystallographic computing program packages such as CCP~ (Ref. 10) and refinement programs such as XPLOR (Ref. 11) that can provide graphica capabilities for map interpretation and structure display; and systematic crystallization protocols’2 and microliter c~stai~ization methods13, which minimize the total number of experiments required to achieve diffraction-quality crystals and which can be adapted to robotic methodsl*. Under suitable conditions, and depending on the project, an individual cycle of SBDD might be completed in as little as a month. The total number of SBDD cycles

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Fig. 2. Example of an X-ray diffraction image. X-ray diffraction data cokcted processed automatically by an area detector system resembles a conventional image. Area detector systems have an expanded dynamic range, sensitivity accuracy over film, in addition to providing immediate on-line detection and processing. Cindy Overby,MolecularStructureCorp.

required for the successful completion of a study will vary from problem to problem but it is not unreasonable to expect 20 or more such cycles in the course of a project. Typically, more than one of these cycles can be executed in parallel, depending on manpower and facilities. A rapid turnover of structure-based design cycles has only been made possible by recent improvements in the individual technologies involved in SBDD. Achievements and challenges Interest in SBDD has stimulated the solution of numerous therapeutically relevant structures by X-ray and NMR methods’*2. These structures have included enzymes, hormones and membrane-associated proteins that have been either solubilized by proteolysis or cloned and expressed without their membrane-anchoring sequences. In some instances, structural information has been applied directly to the design of novel therapeutic agents (e.g. against carbonic anhydrase7 and thrombin15) that are currently

and film and data

undergoing clinical trials; other structures (e.g. HIV-l protease5*“, thymidylate synthetaselb and purine nucleoside phosphorylase’7) are under active investigation by SBDD methods. Structures of related proteins have also served as surrogates for target proteins whose isolation and study have proven difficult (as in the design of angiotensin-converting enzyme inhibitors based on the structures of carboxypeptidase A and thermolysin’s). In these and other instances, SBDD has proven its ability to improve the in vitro binding affinity and potency of lead compounds against their macromolecular targets, both predictably and with regularity’s2. This potency has been achieved by the application of ad hoc modeling strategies, some global principles and, increasingly, automated methods**9. SBDD also has its share of technical challenges. Few structures of integral membrane proteins have yet been solved by crystallography - and none as routinely as soluble enzymes. Furthermore, none of

those solved structures include integral membrane receptors that are known therapeutic targets, such as the biochemically wellcharacterized PI-adrenoce?torJ9. As such, a whole class of impcrtant target macromolecules - strongly represented in the therapeutic agents most used - is currently beyond the reach of the SBDD paradigm. Other challenges include resolution of practical problems in in viva efficacy, toxicity, pharmacokinetics and bioavailability that are beyond direct accessibility through the SBDD approach. These issues are problematic for all approaches to drug discovery, including natural product screening. Success in this area has been achieved empirically and through the co-ordinated and sustained application of intelligent trial-and-error medicinal chemistry, guided by well-designed biochemical and biological assays relevant to the activities being optimized 2o. Nothing intrinsic to the SBDD paradigm precludes its integration into this broader drug development approach, although the technique is often judged in isolation on its (lack of) success. In one case, however, SBDD has been successfully applied to the design of irz viuo topical bioavailability in carbon anhydrase inhibitors for the treatment of ocular hypertension and glaucoma7. In this specialized system, the physiological properties required of topical ocular agents are reasonably well understood, and loose bioavailability design rules have been defined21. SBDD studies of carbonic anhydrase in complex with a series of sulphonamide inhibitors were able to define regions within those lead molecules that were important to binding and in vitro inhibitory potency. These studies also identified other regions that were both accessible and replaceable for achieving the physical and chemical properties required for ill vivo bioavailability. Lead molecules were designed with those bioavailability rules in mind, balancing the hydrophobicity properties needed to penetrate the cornea with the hydrophilicity properties dictated by the requirements of forni?:lation2’ yet maintaining the in vitro POtency needed to effectively inhibit the enzyme7. These studies have now led to a topical carbonic

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FK506

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Fig. 3. Left: chemical structures of the immunosuppressive agents FK566 and rapamycin. Note the similarity between the two in the -rimon regjon is now k~wn to bind to FK506 binding protein ~fK3Pl2~ in both complexes, as tower-teft quarter of $9~ stru@ures. TL., ,ib cyI determined by X-ray diffraction studies. Right: electron density maps of the immu~su~pressant binding region of FKBPIP in complex with FK506 (top) and rapamycin (bottom). From Yamashita, M. et al., unpublished.

anhydrase inhibitor, dorzolamide hydrochloride (USAN) (‘Trusopt’), in Phase III clinical trials by Merck (USA). In other areas, such as in acquired immune deficiency syndrome, unfortunately, a similar success for SBDD has not been forthcoming. An educated guess based on published hterature”*6 would suggest that SO-100 HIV-l protease inhibitors have been synthesized that are sufficiently potent to stop both enzyme activity and viral replication in cell culture in vitro. Many of these have had their structures solved in complex with the protease$ three are in clinical trials, but none is yet a drug. Most are either not absorbed orally or are cleared rapidly from the circulation by the liver and recycled to the digestive system through the bile5. The in-

ability to define parameters to avoid such problems has frustrated attempts to apply SBDD to yield true pharmaceuticals (rather than in vitro inhibitors) for this disease. Application in immunosuppression As outlined elsewhere in this issue (see J. Liu, T. E. Starzl and colleagues, and J-F. Bach) the discovery at Sandoz” in the early 1970s of ciclosporin (initially as an antibiotic) has precipitated a revolution in medicine and biology. Two other immunosuppressants, rapamycin23 and FK506 (Ref. 24) were also discovered as natural product screening leads, with rapamycin being first identified as an antifunga123. JThe striking similarity to FK506 in chemical structure (see Fig. 3) led to a re-examination

of rapamycin as an immtmosuppressive agentz5.] Ciclosporin is on the market and FK506 is in clinical trials. All three agents clearly are effective as immunosuppressants, yet all have severe, though somewhat differing, associated toxicities and unfavourable pharmacokinetic properties26. These shortcomings may be acceptable for a life-or-death indication such as organ transplantation but are less suitable or appropriate for non-acutely fife-threatening applications, such as the control of autowhere the immune diseases, efficacy of these agents has been demonstrated27 but where safety is a significant issue. Due to the complicated macrocyclic structure of these molecules (see Fig. 3) and the large number of stereo centres present, optimizing in zlivo parameters by synthe-

TiPS - May 2993 IV&. 141 sizing analogues has been impossible. From reported de ncwo syntheses of these molecules28~29, a minimum synthesis time is estimated at three or more months per analogue. One can therefore understand why these immunosuppressive agents - though currently in use in patients - remain as the original and unmodified lead molecules discovered by natural products screening. This is rare for a marketed drug or a clinical lead candidate at such an advanced stage of development (other macrocyclic natural products, including antibiotics and anti-neoplastic agents, are additional examples of such pure screening leads as drugs). It is sobering to remember that the therapeutically relevant pathway considered here was first identified - by the discovery and activity of ciclosporin itself through natural product screening of fungal metabolites in soil sample$. The prominence of natural product screening as the principal source of novel structural leads in drug development continues to be reinforced by example (see, for example, Ref. 30). Interest in immunosuppression as a possible target area for the SBDD approach was stimulated by the initial hypothesis that the immunosuppressive properties of agents such as ciclosporin, FIG06 and rapamycin were derived from their ability to inhibit the peptidyl prolyl isomerase (PPIase or rotamase) activity of their respective binding proteins {or immunophilins). Two small but abundant proteins were identified with this rotamase activity: cyclophilin (M, = 14000), which binds to ciclosporin, and FKBP12 (FK506 binding protein; M,= 12000), which binds to FIG06 or rapamycin (Fig. 3). Interestingly, though both proteins share rotamase activity, they demonstrate no obvious sequence31*32 or structura133-35 homology. Rotamase inhibition by analogous inhibitors~ appeared to correlate linearly with the degree of immunosuppression observed with those compounds. These and other observations fostered the conclusion that immunosuppression might be achieved simply by designing rotamase inhibitors superior against a known immunophilin. As discussed above, enzyme in-

Fig. 4. A computerized representation of the FKSOLi-FKBP12-calcineurin-calmodulin complex. FK506 (green) in complex with FKBP 72 (yellow) and es/mod&n (red) are rep~sented by their sotved X-ray s~ucturaf coordinates ff?ef, 25 and Eabu, Y. S. et al. ft988) J. Mol. Bial. 204, Wf-2#41. Catcineurtn 6 (pale blue, small molecule) is represented by the structure oftroponin C [Sakshur, K. .‘.. et al. (1988) J. Biol. Chum. 263, 1626-16471, a Ca”-binding protein WIM sequence homology. Calcineurin A (pale blue, large molecule) is represented by the structure‘ of a protein of a~roximate~ equal molecular weight. Fiiure kindly generated by Jim Griffith.

hibitor projects are preferred in the SBDD approach because they focus on antagonizing an activity (catalysis) for their success and because all the interactions relevant to in vitro potency (inhibition) can be examined within the framework of the solved crystal structures of enzymeinhibitor complexes. We now know that immunophilin-based immunosuppression is considerably more complex than was originally believed and that the scope of the problem remains largely undefined. The complexes of cyclophilin with ciclosporin and of FKBPl2 with FK506 both function as inhibitors of a known Ca”+-dependent phosphatase, calcineurin37‘3s, whose activity leads ultimately to IL-2 mRNA transcription, IL-2 production and the activation of T cells responsible for rejection events25,39. Calcineurin itself is a protein com.p!ex that includes calcineurin A (CNA; M, =60000), calcineurin B (CNB; M, = 2000~), calmodulin and the Ca2+ ions required for activation”O. CNB shows distinct sequence homology with calmodulin and proteins. other Ca2+-bindino These components Oare shown

schematically in Fig. 4. FKBP12 in complex with rapamycin does not bind calcineurin38. The protein receptor for the FKBP12-rapamycin complex is not known. Rapamycin mediates suppression of T-cell activation by inhibiting lymphokine-induced proliferation36, specifically by inhibiting the IL-2-stimulated phosphorylation and subsequent activation of p70 56 kinase4’. In response to this newIy defined complex situation, SBDD can be used to improve the immunosuppressive properties of FK506 or ciclosporin in two ways. One could seek to isolate the calcineurin-immunophilin-immunosuppressant complex, crystallize it and solve its structure as a prelude to applying the SBDD process in the manner described above, albeit to a much larger enzymeinhibitor system than was considered before calcineurin entered the picture. The second approach would ignore the calcineurin structure per se and would assume that by mimicking the complexes of FK506 or ciclosporin with their relative immunophilins in sufficient detail, one would obtain immunosuppression through the calcineurin mechanism. This latter

strategy would require a conceptual advance in the SBDD proLoss, since it would involve, in effect, the more difficult design of an immunophilin-mediated ‘antagonist’ of the phosphatase activity of the cafcineurin ‘r~epmr {and, hence, an ‘agonist’ Of immunosuppressio~~. fn this new version of the standard approach, as applied to the FKSO&FKBP12additional system, calcineurin effort has been necessary in designing agents and determining X-ray structures that probe the retevant conformational features of the structurally unknown caicineurin *receptor‘. Analysis of the chemical struttures of FK506 and rapamycin (Fig. 3) shows common features between the two moiecutes that were thought to mediate their binding to FKBPlZ. This observation has been confirmedM by Xray c~stallographic studies of the FK506 and rapamycin complexes with FKBP12. The remaining structural elements can be thought of as the effector domains of these motecufe8 that promote the differential immunosuppressive effects they express - presumably via their interaction with a corresponding receptor fcalcineurin for FK506 and unknot for rapamyc~n~. Though this has been an attractive and quite useful framework for the SBDD approach, it is difficult nonetheless to attribute such a key role in the mechanism of immunosup pression to the specific effector domain confo~at~ons of what are, after a& fungal metabolites. Sitedirected mutagenesis studies of FKBPU (Ref. 43) support a less radical hypothesis - that these agents act, at least in part, through their effect on the conformation of flexible regions of their binding proteins. Complexes of mutant FKBPlZ with FK506 that fail to inhibit calcineurin phosph~tase activity have been shown to retain their PPIase inhibitory activity43. They have also been shown by Xray crystallographic analysis of the complexes to bind FK506 correctly @oh, S. and Navia, M. A., unpublished). In spite of additional opportunities for therapeutic intervention implicit in the more complex mechanisms emerging from a more detailed study of immunosuppression, the interactions of immunosupp~ssant with im-

Fig. 5. Size distributjon and comparison of immunological mofeculee of therapeutic interest. {a) Cartoon of the complement component Ctq represented to scale as determined by ei~~mn mi~ro~py lTschopp, J. et al. ~f93~~ fur, J. lmmunol. 10, 523-535~~ (b) Intact human myefoma immunoglobu~i~ G [Siiverffan, E. W. et al. (1977) Proc. Natl Acad. Sci. USA 74, 574~51441. The complement system is activated by the interaction of these molecules of fadicaiiy different size. (c) Human &4-B27 class I ~s~~om~a~b~lity antigen [Maddens D. l?. et al. f793~~ Nature 353, 321-325], an example of a rne~~e-a~~~o~~ profein ~u~ljzed by sefective proteo&sis. (d) Ceil-s&ace g~#p~u~in CD4 Wang, J. H. et al. f199Oj Nature 348, 47?-4fBf expressed as a soluble protein without membrane-anchoring sequence. CD4 is involved in T-cell activation and in binding of H/V virus. (e) Thr? lymphokine lumof necrosis factor [Eck, M. J. and Sprang, S. I?. (7969) J. FM. Chem. 2% 77595-I 7605]. (f) immunosuppressan~ binding protein FKBP12 with FK506 nearby. Scaie bar = fO nm.

munophihn may still be the choice target for SBDD. First, ciclosporin, FK506 and rapamyein are being used in the clinic, albeit with some serious side-effects and pha~acokinet~c complications. Other ‘known’ targets in the chain of events leading to immunosuppression may possess still unknown, and perhaps more severe, side-effects of their own. Further, a considerabfe body of understanding - from the molecutar to the clinical level has been developed for immunophilinmediated immunosup~~ssion. That level of knowledge would have to be developed for other targets. Finally, these molecules exert their effects on immunosuppression early in the process, enabling a small absolute dose of agent to have an amplified influence on the desired end effect.

The complexity of immunological processes makes their exploitation a difficult arena for SBDD at its present stage of de-

velopment. The autodestructive potential associated with immunemediated effects means that they are often under multiple levels of interconnected regulation. Structure-function analysis by sitedirected mutagenesis or Iigand displacement suggests that the interactions between molecules can be complicated and subtle. Nonetheless, a number of structures of therapeutic relevance to immunology (see Fig. 5) have been solved by X-ray diffraction and NMR techniques and could be used to initiate drug development based on the SBDD paradigm. Many of these solved structures are of isolated effector proteins (e.g. interieukins and interferon& where an effector must interact with its target receptor molecule to produce the observed biological activity. SBDD based on effector motecdes in isolation may be p~blematic. Histo~ca~Iy~ for example, structural information on native and mutant insulin effectors, in association with a wealth of structure-guided biochemical

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and biological research44, has yet to produce a therapeutically viable small-molecular-weight mimic of insulin activity. Where proteinprotein interactions have been directly observed in atomic detail (as in the structure of the thrombin-hi~din complex45), they can be described as an aggregate of weak intermolecular forces spread over a large area, often involving multiple and locally unconnected polypeptide residues - a difficult template to fill with a small structure. Immunosuppressantimmunophilin complexes may represent an intermediate level of complexity and difficulty, since only the smaller, connected and more compact drug component of the complex need be mimicked effectively to achieve ‘agonist’ activity. Further, many receptors of interest in immunology (and elsewhere) are integral membrane proteins with structures, as discussed above, not routinely solvable by current methods; as such, they remain universally out of reach of SBDD. Fortunately, however, many other receptors of immunological interest are only anchored to the membrane. This means that binding domains can be disassociated and solubilized by selective proteolysis or by expression of suitably truncated recombinant proteins. Examples of these include the T-cell surface CD4 (Refs 46, 47) and CD2 (Ref. 48) receptors and the histocompatibility antigens; the structure of the latter, with their peptide ligands bound, has been solved*9,s0. Crystallization has been reported as a prelude to X-ray diffraction studies for bovine growth hormone5* and tumor necrosis factors2 in complex with their respective receptors. Finally, solubilized progress is clearly evident in the design of low-mole~lar-weight peptidomimetics of some of the smaller peptide hormones, including cholecystokinin analogues such as MK329 (Ref. 53) and cyclic analogues of somatostatin53. The good news is that structural and chemical methods are being marshalled for creditable assault on the synthesis of specific agents to interact with these systems. Ironically, the bad news is that we have yet to achieve sufficient understanding of many of these systems to identifv the aooroariate tareets

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and to dissect fully the consequences of disabling or hyperenabling them. q

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Enzyme inhibition remains the most tractable category of target opportunities for SBDD - both from the technical perspective of analysis by structural methods and from the biological perspective of defining meaningful assays to monitor the process. As such, a focused search for enzyme-based targets for drug design in immunology might prove fruitful in the short term. Identification of the IL-1 converting enzyme as a suitable SBDD target is one such development, and could provide therapeutic inroads into the control of chronic inflammatory diseases, septic shock and other IL-Q-dependent diseases. Acknowledgements We thank our colleagues Joshua Boger, Matthew Harding, Mark Murcko, Keith Wilson, Sergio Rotstein, I-Iector Gomez and Vicki Sato for their continuing assistance and support and for reading this manuscript. References 1 Walkinshaw,

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