Structure-based drug design: applications in immunopharmacology and immunosuppression

Structure-based drug design: applications in immunopharmacology and immunosuppression

immunomodulation Structure-baseddrug design: applications in immunopharmacology and immunosuppression Manuel A. Navia and Debra A. Peattie Structure-...

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immunomodulation

Structure-baseddrug design: applications in immunopharmacology and immunosuppression Manuel A. Navia and Debra A. Peattie Structure-based drug design (SBDD) combines the power of many scientific disciplines, such as X-ray crystallography, nuclear magneuc resonance, medicinal chemistry, molecular modeling, biology, enzymology and biochemistry, in a functional paradigm of drug development. The current strength 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 achievements and challenges in immunopbarmacology, particularly as these 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 information 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 drug-discovery disciplines. A drug development campaign centered on SBDD is likely to consist of a series of tactical extrapolations from experimentally deter,~ou.,~;I--a: . . to. .the. .b y l l H lL__~ .minoA . . . . . . . . . . . . . .err,,,*ra,ral . ~ s li"fo'mation, ix~ xs t~ilS 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 inhibitors 2 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 - much like a piece of gum in a fine watch. This is a much less formidable task than that reqaired 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.

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 determination by X-ray diffraction methods. For nuclear magnetic resonance (NMR) studies of drugtarget interactions in solution, isotope labeling of the protein or drug may be necessary in order to obtain struct~]ral 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 systems) and whole-animal/organism studies in vivo (e.g. efficacy, Initiating the design process pharmacokinetics, toxicity, etc.). The SBDD paradigm is summarized schematically in Finally, it is desirable to identify early in the initial Fig. 1. It consists of an initial phase in which the necess- stage a prototype chemical lead (or 'proto-lead') to ary technical components of the process are mar- structurally delineate the active site or binding region shalled, and a production phase in which those compo- of the target macromolecule and to locate a subset of nents are applied in cyclic fashion. As part of the initial critical interactions therein. In practice, this is not as phase, the desired ,nacromolecular 'drug target' must unreasonable as it might seem since the therapeutic © 1993, Elsevier Science Publishers Lid, IlK. 0167.56991931506.00

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immunomodulation relevance of target macromolecules often stems from such a proto-lead interaction in the first place (for example peptidyl snake venom proto-leads were critical in developing angiotensin-converting enzyme inhibitors) 3. In some circumstances, a proto-lead can be designed by modification of a known natural effector or substrate (for example peptidyl inhibitors of renin 4 and human immunodeficiency virus (HIV-1) protease s,6 were first proposed from a knowledge of the structure of substrates of those enzymes). At times, an existing drug can serve as a proto-lead if its properties can be significantly improved. Topical carbonic anhydrase inhibitors, for example, might substantially improve the treatment of ocular hypertension and glaucoma 7 in comparison to existing systemic agents. Even in situations where suitable proto-leads cannot be identified, computational model-building procedures should be able to jump-start the SBDD process via de novo design of proto-leads based on the structure of the target molecule's surface s,9.

Execution of 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 SI3DD, 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 light 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 me earnest posslole point. pracncal concerns might include ease of synthesis, process scalability, 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 determination by X-ray or NMR methods and are quickly re-examined to determine which of the operating assumptions about the interaction with the drug target molecule require(s) adjustment. If a predicted compound works as expected, one can proceed directly to further model building and synthesis without necessarily confirming the prediction by a structure determination. Even in those situations, however, it is still prudent to check p~riodically by experiment the validity of the assumptions underlying 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 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 reliability and efficacy of assay procedures used. ~L

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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 regioand 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, ab initio calculations and the implementation of largescale conformational searches in the de novo design ,o¢ drug candidate molecules. Perhaps the most remarkable technological advance is the evolution of X-ray crystallography and NMR to the status of applied science~. In crystallography, 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 film methods (see Fig. 2); high-performance workstations that can execute general crystallographic computing program packages such as cop4 (Ref. 10) and refinement programs such as XPLOR (Ref. 11); that can provide graphical capabilities for map interpretation and structure display; and systematic crystallization protocols n and microliter crystallization methods 13,which minimize

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F~ 2. X-ray diffractiondata collectedand processedautomaticallyby an area detector system resembles a conventional film image. Area detector systems have an expanded dynamic range, sensitivity and accuracy over film in addition to providing immediate on-line detection and data processing (Cindy Overby, MolecularStructure Corp.).

the total number of experiments required to achieve diffraction-quality crystals and which can be adapted to robotic methods 14. 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 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 structurebased 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 ~. 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 anhydrase ~ and thrombin *s) that are currently undergoing clinical trials; other structures (e.g. HIV-1 protease s,6 thymidlate synthetase ~6 and purine nucleoside phosphorylase) 17 are under active investigation by SBDD methods. Structures of related proteins have also served as surrogates for target proteins whose isolation and study has

Immunolo~ Today

proven difficult (as in the design of angiotensin-convetting enzyme /nhibitors based on the structures of carboxypeptidase A and ~hermolysin) Is. 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 l,z. This potency has been achieved by the application of ad hoe modeling strategies, some global prindples and, increasingly, automated methods s'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 well-characterized ~,-adrenoceptor Is. As such, a whole class of important 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 vivo 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 coordinated and sustained application of intelligent trial-and-error medicinal chemistry, guided by well-designed biochemical and biological assays relevant to the activities being optimized 2°. 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 in vivo topical bioavai!abi!ity in carbonic anhydrase inhibitors for the treatment of ocular hypertension and glaucoma 7. In this specialized system, the physiological properties required of topical ocular agents are reasonably well understood, and loose bioavailability design rules have been defined2k SBDD studies of carbonic anhydrase in complex with a series of sulfonamide 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 in 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 formulation z* yet maintaining the in vitro potency needed to effectively inhibit the enzyme7, These studies have now led to a topical carbonic 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 the published literatures,6 would suggest that 50-100 HW-1 protease inhibitors have been synthesized that are sufficiently potent to stop both enzyme activity and

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lmm..olo~ Today

identified as an antifungaF 3. (The striking similarity to FK506 in chemical structure (see Fig. 3) led to a reexamination of rapamycin as an immunosuppressive agent25.) Cyclosporin 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 unfavorable pharmacokinetic properties 26. These shortcomings may be acceptable for a life-or-death indication such as organ transplantation but are less suitable or appropriate for non-acutely life-threatening applications, such as the control of autoimmune diseases, where the efficacy of these agents has been demonstrated 27 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 centers present, optimizing in vivo parameters by synthesizing analogs has been impossible. From reported de novo syntheses of these molecules 2sag, a

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Fig. 4. A computerize./ .,presentation of the FKSO6--FKBP12-calcineurincalmodulin complex, a) FK506 (green) it', complex with b} FKBP12 O,ellow) and c) calmodulin (red) are represented by their solved X-ray structural coordinates IRe[. 25 and Babu, ¥.S. et al. (1988) J. Mol. Biol. 204, 191-204]. d} Caicineurin B (pale blue, large molecule) is represented by tbe structure of troponin C [Satysbur, K.A. et al. (1988) J. Biol. Chem. 263, 1628-1647], a Ca2÷-bindingprotein witb sequence homology, e) Calcineurin A (pale blue, small molecule) is represented bv tbe structure of a protein of approximately equal molecular weigbt. Figure kindly provided by Jim Griffitb.

be achieved simply by designing superior rotamase inhibitors against a known immunophilin. As discussed above, enzyme inhibitor 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 enzyme-inhibitor 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 cyclosporin and of FKBP12 with FKS06 both function as inhibitors of a known Ca 2÷dependent phosphatase, calcineurin 37,3s, whose activity leads ultimately to IL-2 mRNA transcription, IL-2 production and the activation of T lymphocytes responsible for rejection events2sjg. Calcineurin itself is a protein complex that includes a calcineurin A (CNA; M~=60 000), calcineurin B (CNB; M~=20 000), calmodulin and the Ca z÷ ions required for activation4°. CNB shows distinct sequence homology with calmodulin and other CaZ*-binding proteins. These components are shown schematically in Fig. 4. FKBP12 in complex with rapamycin does not bind calcineurin 3s. The protein receptor for the FKBP12-rapamycin complex is not known. Rapamycin mediates suppression of T-cell activation by inhibiting lymphokine-induced proliferation 36, specifically by inhibiting the IL-2-stimulated phosphorylation and subsequent activation of p70 $6 kinase41. In response to this newly defined complex situation, SBDD can be used to improve the immunosuppressive properties of FK506 or cyclosporin in two ways. One could seek to isolate the calcineurin-immunophilinimmunosuppressant complex, crystallize it and solve

minimum synthesis time is estimated at three or more months per analog. 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 r~l,,A~ ~..~,~,-.I..;.... the c ~ r x r x ir~ ~rrn~'rnro a e a p.~.,.,.~ ,., -vv.-,~ examples of such pure screening leads as drugs)° It is sobering to remember that the therapeutically in the manner described above, albeit to a much larger relevant pathway considered here was first identified - enzyme-inhibitor system than was considered before by the discovery and activity of cyclosporin itself - calcineurin entered the picture. The second approach through natural product screening of fungal metab- would ignore the calcineurin structure per se and olites in soil samples2z. The prominence of natural prod- would assume that by mimicking the complexes of uct screening as the principal source of novel structural FK506 or cyclosporin with their relative immunoleads in drug development continues to be reinforced phi!ins in sufficient detail, one would obtain immunoby example (see, for example, Ref. 30). Interest in suppression through the calcineurin mechanism. This immunosuppression as a possible target area for the latter strategy would require a conceptual advance in SBDD approach was stimulated by the initial hypoth- the SBDD process, since it would involve, in effect, the esis that the immunosuppressive properties of agents more difficult design of an immunophilin-mediated such as cyclosporin, FK506 and rapamycin were 'antagonist' of the phosphatase activity of the calderived from their ability to inhibit the peptidyl prolyl cinearin 'receptor' (and, hence, an 'agonist' of isomerase (PPlase or rotamase) activity of their re- immunosuppression). In this new version of the stanspective binding proteins (or immunophilins). Two dard approach, as applied to the FK506-FKBP12small but abundant proteins were identified with this calcineurin system, additional effort has been necessary rotamase activity: cyclophilin (M~:14 000), which binds in designing agents and determining X-ray structures to cyclosporin, and FKBP12 (FK506 binding protein; that probe the relevant conforrnational features of the Mr=12 000), which binds to FK506 or rapamycin (Fig. structurally unknown calcineucin 'receptor'. 3). Interestingly, though both proteins share rotamase Analysis of the chemical structures of FK506 and activity, they demonstrate no obvious sequence 31,3z or rapamycin (Fig. 3) shows common features between structural 33-3s homology. Rotamase inhibition by the two molecules that were thought to mediate their analogous inhibitors36 appeared to correlate linearly binding to FKBP12. This observation has been conwith the degree of immunosuppression observed with firmed 34 by X-ray crystallographic studies of the those compounds. These and other observations FK506 and rapamycin complexes with FKBP12. The fostered the conclusion that immunosuppression might remaining structural elements can be thought of as the

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immunomodulation effector domains of these molecules42 that promote the differential immunosuppressive effects they express presumably via their interaction with a corresponding receptor (calcineurin for FK506 and unknown for rapamycin). 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 immunosuppression to the specific effector domain conformations of what are, after all, fungal metabolites. Site-directed mutagenesis studies of FKBP12 (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 FKBP12 with FK506 that fail to inhibit calcineurin phosphatase activity have been shown to retain their PPIase inhibitory activity43. They have also been shown by X-ray crystallographic analysis of the complexes to bind FK506 correctly (S. Itoh and M.A. Navia, 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 immunosuppressant with immunophilin may still be the choice target for SBDD. First, cyclosporin, FKS06 and rapamycin are being used in the clinic, albeit with some serious side-effects and pharmacokinetic 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 considerable body of understanding - from the molecular through to the clinical level - has been developed for immunophilin-mediated immunosuppression. That level of knowledge would have to be developed for other targets. Finally, these molecules exert their ^££^^,.~ g:ll~t,,t3

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enabling a small absolute dose of agent to have an amplified influence on the desired end effect. Role in immunology The complexity of immunological processes makes their exploitation a difficuk arena for SBDD at its present stage of development. The autodestructive potential associated with immune-mediated effects means that they are often under multiple levels of interconnected regulation. Structure-function analysis by sitedirected mutagenesis or ligand displacement suggests that the interactions between molecules can be complicated and subtle. Nonetheless, a number of structures of therapeutic relevance to immunology (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. interleukins and interferons), where an effector must interact with its target receptor molecule to produce the observed biological activity. SBDD based on effector molecules in isolation may be problematic. Historically, for example, structural information on native and mutant insulin effectors, in association with a wealth of structure-guided biochemical and biological research44, has yet to produce a therapeuti-

Immunology Today

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Fig. 5. Size distribution and comparison of immunological molecules of tberapeutic intere.t, a) Cartoon of tbe complement component Clq represented to scale as determined by electron microscopy [Tscbopp, J. et al. (1980) Eur. J. Immunol. 10, $29-$3S]. b) Intact buman myeioma immunoglobulin lgG [Silverton, E.W. et al. (1977) Proc. Natl Acad.Sci.USA74, 5140-5144]. The complement system is activated by the interaction of these molecules of radically different size. c) Human HLA-B27 class I bistocompatibility antigen [Madden, D.R. et al. (1991) Nature 3S3, 321-32S], an example of a membrane-ancbored protein solubilized by selective proteolysis, d) Cell-surface glycoprotein CD4 [Wang, J.H. et ai. (1990) Nature 348, 411--418], expressed as a soluble protein without membrane-ancboring sequence. CD4 is involved in T-cell activation and in binding of HIV virus, e) The lympbokine tumor necrosis factor [Eck, M.]. and Sprang, S.R. (1989) J. Biol. Chem. 264, 17S95-17605]. D lmmunosuppressant binding protein FKBP12 with FKS06 nearby. Scale bar = 10 nm. cally viable small-molecular-weight mimic of insulin activity. Where protein-protein interactions have been mrecuy ooserveu m atomic aetau ~as in the structure of the thrombin-hirudin complex4S), 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' zctivity. 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 proteolvsis 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 solved49's°. Crystallization has been reported as a prelude to X-ray diffraction studies for bovine growth hormone 51 and tumor necrosis factor s2 in complex with their respective

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immuno modulation solubilized receptors. Finally, progress is deafly evident in the design of low-molecular-weight peptidomimetics of some of the smaller peptide hormones, including cholecystokinin analogs such as MK329 (Ref. 53) and cyclic analogs of somatostatin s3. 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 identify the appropriate targets and to dissect fully the consequences of disabling or hyper-enabling them.

Crystallization of Nucleic Acids and Proteins: A Practical Approach (Ducruix, A. and Gie#, R., eds), pp. 291-310,

IRL Press 1S Banner,D.W. and Hadvary, P. (1991) J. Biol. Chem. 266, 20085-20093 16 Reich, S.H. etal. (1991)I. Med. Chem. 35, 847-858 17 Montgomery,J.A. et al. (1993) ]. Med. Chem. 36, 55-69

18 Monzingo,A.F. and Matthews, B.W. (1984) Biochemistry 23, 5724--5729 19 Dixon, R.A.F. etal. (1986) Nature 321, 75-79 20 Humphrey,M.J. and Smith, D.A. (1992) Xenobiotica

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